WO1999063347A2 - Procedes, instruments et marqueurs relatifs a des essais electrochimiques - Google Patents

Procedes, instruments et marqueurs relatifs a des essais electrochimiques Download PDF

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Publication number
WO1999063347A2
WO1999063347A2 PCT/EP1999/003756 EP9903756W WO9963347A2 WO 1999063347 A2 WO1999063347 A2 WO 1999063347A2 EP 9903756 W EP9903756 W EP 9903756W WO 9963347 A2 WO9963347 A2 WO 9963347A2
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conducting
semi
structures
wherem
assay
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PCT/EP1999/003756
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English (en)
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WO1999063347A3 (fr
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Mark Howard Jones
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Mark Howard Jones
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Priority to AU43721/99A priority Critical patent/AU4372199A/en
Priority to EP99926483A priority patent/EP1093584A2/fr
Publication of WO1999063347A2 publication Critical patent/WO1999063347A2/fr
Publication of WO1999063347A3 publication Critical patent/WO1999063347A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • This invention relates to an instrument, a method and labels for carrying out assavs using an electrochemical reaction on a conducting or semi-conducting material in contact with an electrolyte but not in electronic contact with an external electronic circuit.
  • the molecules are radiolabeled and are detected by autoradiography
  • the analytes mav also be transferred to a membrane and analysed after they have been bound. Examples of this are hybridizations southern, northern, and lmmunoblomng westerns These methods are typically time consuming and/or lack sensitivity
  • filter wicking methods These are used for various analytes such as glucose, cholesterol, drugs, antigens, antibodies and nucleic a ⁇ ds
  • antibodies these are called lmmunochromatographic assays
  • filter strip, wicking methods or lateral-flow offer the beauty of simplicity All that is necessary is to apply a sample, for example blood, urine or saliva, and this results in the dned and labelled antibody dissolving and mixing with the sample and starting the an ⁇ bodv/analvte binding reaction During this binding reaction the sample and labelled antibody are wicked (via capillary flow) along a strip which at a predetermined point contains an immobilised second antibody against the analyte of interest.
  • the analyte binds to the antibody.
  • This immune complex then at some point is transported in the liquid flow to the capture antibody which is immobilised at a given site within the filter or supporting phase.
  • the analyte-labelled antibody complex interacts with the immobilised capture antibody it is captured by binding to an alternative epitope on the analyte of interest resulting in the immobilisation of the label which is attached to the labelled antibody and complexed with the analyte.
  • the fluid continues to wick or flow past the immobilised antibody carrying any un-complexed (not bound to analyte of interest) labelled antibody away from the immobilised capture antibody.
  • the labelled antibody is detected.
  • these assays use a number of different labelling methods such as particles and enzymes.
  • additional buffer and/or solutions can be added to give other types of signal and improve the binding reactions.
  • the sample is added and allowed to incubate. This is then followed by a wash, and the second binding reagent (labelled) is added followed by a wash.
  • the substrate is then added to detect the bound labelled reagent.
  • Electrochemical-based sensors for the detection of biomolecules have been developed into highly successful commercial products. This has been exemplified by the measurement of glucose based on the detection of electrical changes that occur during an electrochemical reaction.
  • Current glucose detectors use an enzyme such as glucose oxidase to provide the selective recognition and generation of a detectable signal by an electrode.
  • Some of these products such as the system manufactured by Boehrmger Mannheim, use direct electrochemical oxidation of hydrogen peroxide produced by the enzyme.
  • MediSense Inc. uses a modified ferrocene as a redox mediator between the enzyme and the electrode. This removes problems due to oxygen concentraoons and pH (M.G. Boutelle et al. J MoL Rec. 1996.
  • Electrochemical technology has also been applied to the development of electrochemical methods based on the genera ⁇ on of light as a signal (Igen lnc, Gaithersburg MD).
  • a specific laObel, typically Ru is used attached to a binding species through a spacer arm and the capture of this label is detected by the generauon of ex ⁇ ted-state Ru (2,2-b ⁇ py ⁇ dyl) 3 2+ via a redox reaction at the surface of an electrode connected via an electromc circuit to a potendostat.
  • the voltage is controlled at the surface of the externally, electronically connected electrodes within the electrochemical cell to surface potentials less than 3 volts due to the nature of the instrument and the electrochemistry of this system.
  • This electrochemistry is conducted using a potenuostat to control the potential at a working electrode.
  • These instruments for controlling electrochemistrv are designed to apply voltages of less than 10 volts and electrochemical methods have thus been restricted to these low voltages applied to an externally, electronically connected electrode surface.
  • luminol and its denva ⁇ ves can be subjected to electrochemical excitation resulting in the emission of light.
  • the use of the luminol system typically involves combination with a peroxidase-producing enzyme which generates hydrogen peroxide for the activation of the luminol light emission on electrochemical activation.
  • electrochemistry for assaying biomolecules based on the detection of specific biomolecules directly via electrochemical methods or via the use of enzymes or other biocatalysts used to generate detectable species or via the use of electrochemiluminescent labels as in the case of the ruthenium tnsbipy ⁇ dyl label used by Igen me, Gaithersburg MD (WO 86/02734).
  • the essential element of all these methods has been the need to bring the reagents and/or binding species in close proximity to an electrode surface which is in direct electronic contact via wires or equivalent electron conduction material to an external control electromc circuit.
  • the electrode systems in these cases are typically made of a three-electrode system with working, counter and reference electrodes in contact with an electrolyte that contains the sample. These three electrodes are then in electromc contact outside the sample or electrolyte chamber via wire or equivalent electron conducting material to a potenttostat This configuration of electrodes linked via a potendostat allows precise control of the voltages at the working electrode.
  • the electrode configuration is made up of two electrodes and the counter and reference electrode are combined with some success.
  • electrochemical-based assay systems have been designed using electrodes that are exposed to an electrolyte containing the sample and connected by external contacts via wire or equivalent electron conducting material to a potendostat
  • electrochemical assay systems make use of measurable electrochemistry, for example, by measuring electromc properties or light generated at the surface of these direcdy controlled and connected electrodes.
  • These systems suffer from the problem of not being able to detect and analyse the presence of analytes in the bulk solution. If they are able to effectively capture the analytes in bulk solution, e.g. using beads, then these beads need to be captured or otherwise brought to the surface of an externally, electronically connected electrode.
  • the electrolyte contains a suspension of conducting or semi-conducting particles that are subjected to an electrical field. This results in the generation of anodic and cathodic faces on the conducting particles and results in electrochemical reactions at the surface of the conducting particles.
  • These methods have not been used to generate a detectable signal or material which is related to a binding event or the presence of a molecule or analvte of interest for the purpose of determining the presence or concentration of these molecules or analytes in a sample
  • the present invention provides for the application of bipolar electrochemistry in a new way for diagnostic applications and constitutes an improvement over those currently known Various methods and examples are disclosed.
  • the level of detectable signal or material being detected is related to the amount or presence of the molecules or analytes of interest
  • the invention is an improvement over the existing art in that it allows the electrochemical reaction to occur more effectively m the bulk solution providing for more rapid assays of greater sensitivity. It is a further improvement in that it provides a means for assaying multiple analytes in an array without the need to provide multiple electrode contacts or large extended electrode surfaces. Also the invention is a method for generating light using a bipolar electrode not in electromc contact with an external electromc circuit.
  • This invention includes an assay method for an analyte of interest using an electrochemical process
  • the assay compnses the establishment of contact between the sample to be assayed for an analvte of interest and at least one structure of conducting and/or semi-conducting material not in electromc contact with an external electromc circuit; followed by the application of a voltage gradient across at least a portion of at least one structure of conducting and/or semi-conducting material not in electromc contact with an external electromc circuit, the voltage gradient bemg such that at least one electrochemical reaction takes place on at least one of at least one structure of conducting and/or semi-conducting material not in electromc contact with an external electromc circuit; then the detection and/or quantita ⁇ on of at least one electrochemical reaction directly and/or indirecdv and determination of the presence and/or quantity of the analyte of interest Variations are possible in this assay, for example, the structure of conducting and/or semiconducting material coupled to a binding species,
  • the assay method as just descnbed above in a selected embodiment includes use of additional binding species such as those free m solution and/or coupled to electrochemically detectable spe ⁇ es which include compounds able to support electroche ⁇ uluminescence or chemiluminescence.
  • spe ⁇ es include, for example- antibody, antigen, nucleic a ⁇ d, receptor, kgand, lectin, nucleic a ⁇ d analogue, streptavidin, avidin, biotin, lmminobiotin, aminobiotin, digoxin, fluorescein.
  • the use of various combinations of binding spe ⁇ es is also contemplated to allow for a 'universal' capture, including the use of immobilized steptavidin with a se ⁇ es of biotinylated antibodies.
  • the structure of conducting and/or semiconducting material includes numerous materials depending on the desired chemist ⁇ es, examples include: carbon, gold and platinum, graphitic carbon fibres, fibrils or nanotubes, graphitic particles, glassy carbon particles, carbon films, sputtered carbon, carbon pastes and carbon composites.
  • This invention is also an assay where a sample has added to it an exogenous enzyme for a substrate in a sample or has added to it an exogenous substrate for an enzyme in a sample to form a mixture of an enzyme with a substrate
  • This mixture can either be formed before or after contacting a sample with a structure of conducting and/or semi-conducting material not m electromc contact with an external electromc circuit, to form a mixture.
  • a voltage gradient is applied across the mixture, the voltage gradient being such that an electrochemical reaction takes place on the structure of conducting and/or semiconducting material not in electromc contact with an external electromc circuit, wherein an electrochemical reaction occurs with a product of the enzyme or the exogenous enzyme action, either direcdy or lndirecdy.
  • the electrochemical reaction is detected and/or quanbtated direcdy or lndirecdy, allowing the determination of the presence or quantity of the substrate or the enzyme in the sample.
  • enzymes and substrates include those involved m dehydrogenatton and/or oxidase enzyme reactions.
  • electrochemistry include those linked to electrochemiluminescence or chemiluminescence.
  • This invention is also an apparatus for the detection or assay of an analyte and comp ⁇ ses: an elect ⁇ cal power supply able to deliver a voltage; a cell able to accept a sample containing structures of conducting and/or semi-conducting material, wherein the cell contains at least two electrodes which are externally and electromcally connected to the elect ⁇ cal power supply and the cell is either an integral part of the apparatus or is a removable element
  • the instrument also includes a detector able to detect electrochemistry and is selected from the following film, photomul ⁇ plier tube, photodiode, phototransistor, CCD camera, electrochemical cell, wherein the detector is selected and/or placed such that it optimally detects electrochemistry from structures of conducting and/or semi-conducting material and avoids detection of electrochemistry from the two electrodes which are externally and electromcaUv connected to the elect ⁇ cal power supply.
  • An example of a cell comp ⁇ ses at least three functional zones- a central zone for the sample to be assayed and at least two zones which flank the central zone and which contain the two electrodes wherein each electrode of the two electrodes is contained within each of the two zones which flank the central zone.
  • the central zone can be separated from the flanking zones by a membrane which is a semi-permeable membrane.
  • the central zone can have a window for the detector allowing solely a view of the central zone.
  • the apparatus can be controlled by va ⁇ ous means for example by a computer or microprocessor.
  • kits for performing an assay comp ⁇ sing a bmding spe ⁇ es labelled with an electrochemically detectable spe ⁇ es and a structure of conducting and/or semi-conducting material coupled to a second bmding spe ⁇ es, wherein the structure of conducting or semi-conducting material not in elect ⁇ cal contact with an external electromc circuit is subjected to an applied voltage in an electrolyte causing electrochemistry to occur on the structure of conducting or semi-conducnng material.
  • kits in one example also make use of a bifuncQonal binding spe ⁇ es which would allow the bmding between structures to generate larger structures of conducting and/or semi-conducting material.
  • the binding between structures could also generate interfaces which would allow coupling of the electrochemistry occurring on the surfaces of two proximal structures. Examples of a detectable electrochemical reaction are those generating light either direcdy or lndirecdy.
  • kits for performing an assay wherein electrochemistry is detected direcdy or lndirecdy from at least one structure of conducting and/or semi-conducting material consisting essentially of; an enzyme or substrate of an enzyme and at least one structure of conducting and/or semiconducting material, wherein the structure of conducting and/or semi-conducnng material is capable of being stimulated to carry out electrochemistry on application of a voltage to an electrolyte in contact with the structure of conducting or semi-conducting material when not in electromc contact with an external electromc control circuit
  • This invention also concerns an immunochromatographic device comp ⁇ smg a bmding spe ⁇ es labelled with an electrochemically detectable spe ⁇ es and a binding spe ⁇ es immobilised, and a solid phase non-conducting material containing at least one structure of conducting and/or semi-conducting material positioned such that electromc connection to an outside elect ⁇ cal circuit can be avoided, wherein the solid phase is not rendered conducting and/ or semi-
  • two electrodes are able to make electromc contact with an external electromc circuit but do not make electromc contact with the solid phase.
  • This invention also concerns the generation of electromagnetic radiation by application of a voltage gradient across at least one structure of conducting and/or semi-conducting material not in electromc contact with an external electromc ⁇ rcuit, the voltage gradient bemg such that at least one electrochemical reaction takes place on the structure of conducting and/or semi-conducting material not in electromc contact with an external electromc circuit wherein the one electrochemical reaction results in the emission of electromagnetic radiation, either direcdy or lndirecdy.
  • An electrocherniluminescent reaction is one example of an electrochemical reaction
  • stabilised dioxetanes or other chemiluminescent molecules can be activated by electrochemistry in such systems.
  • This invention also concerns the generation of electromagnetic radiation within a separations medium and comp ⁇ ses the application of a voltage to a separations medium which contains or is constructed from structures of conducting and/ or semi-conducting material such that the separations medium is not rendered conducting or semi-conducting.
  • separations media include the following materials, styrene, dextran, agarose gels, polyacrylamide gels, cellulose fibres, and de ⁇ vaaves thereof. Examples by which electromagnetic radiation is generated include electrochemiluminescence or chemiluminescence.
  • This invention also concerns an assay for an analyte of interest using an electrochemical process, the assay comp ⁇ smg the following.
  • a voltage gradient is applied across the complex of at least two structures of conducting and/or semi-conducting material not in electromc contact with an external electromc circuit, the voltage gradient being such that an electrochemical reaction takes place on the complex of at least two of the structures of conducting and/or semi-conducting material not in electromc contact with an external electromc errant
  • the detection or quantitation of the electrochemical reactions direcdy or lndirecdy is then followed by the detection or quantitation of the electrochemical reactions direcdy or lndirecdy, and the determination of the presence or quantity of the analyte of interest.
  • This invention also concerns an assay for an analyte of interest using an electrochemical process, the assay composing the following Contact is established between a sample to be assayed and a structure coated or otherwise linked to a selected compound such that an analyte interacts enzyma ⁇ callv or by bmding with the structure via the selected compound resulting in a modified structure.
  • This is followed bv establishing contact between the previous mixture and a binding spe ⁇ es able to bind to the modified structure to form a second modified structure, wherein the bmding spe ⁇ es is linked or labelled with an electrochemically detectable spe ⁇ es.
  • Examples of these structures include polymer base beads coated with at least two binding spe ⁇ es, one spe ⁇ fic for the analyte and one spe ⁇ fic for a complementary binding spe ⁇ es on the structures of conducting and/or semi-conducting material, and the structures of conducting or semiconducting material are coated with the complementary binding species
  • This invention also concerns an assay for an analyte of interest using an electrochemical process, the assay comp ⁇ smg the following. Contact is established between a sample and a structure coated or otherwise linked to a selected compound wherein an analyte interacts with the structure via the selected compound.
  • the structures in one embodiment are bound to both an electrochemically detectable spe ⁇ es and to a bmding spe ⁇ es or enzyme substrate and the structures of conducting and/or semi-conducting material are bound to a bmding spe ⁇ es or enzyme substrate.
  • This invention relates to electrochemical-based assay systems for analytes of interest such as molecules, ions, amme-contaimng molecules, enzymes, substrates, antibodies, antigens, nucleic a ⁇ ds, receptors, kgands, hormones, biomolecules and any combmation of bmding spe ⁇ es which, in operation, incorporate va ⁇ ous structures of conducting or semi-conducting material. These va ⁇ ous structures are not in electromc contact via a wire or equivalent electron conducting media to an external voltage supply
  • the material of the subject invention is conducting or semi-conducting and acts as an electrode which has both an anodic and cathodic face under an applied elect ⁇ c field. Thus, the matenal becomes bipolar with respect to the developed elect ⁇ cal potential.
  • the matenal is m elect ⁇ cal contact via an electrolyte providing an ionic but not an electromc contact to an external electromc circuit
  • the matenal is stimulated to carry out the desired electrochemical reactions by applying a voltage gradient across at least a portion of the electrolyte which is in contact with the conducting or semi-conducting material. This results in the development of anodic and cathodic portions on the conducting or semi-conducnng matenal surface such that electrochemistry occurs on at least a portion of the anodic or cathodic portions of the conducting or semi-conducting material in contact with the electrolyte.
  • the result of the electrochemical reaction is to produce a detectable signal (such as light) or to produce a detectable molecular spe ⁇ es (such as a chromophore or fluorophore or electrochemically-reacQve molecule) which allows the detection and/or quanota ⁇ on of the analytes of interest
  • a detectable signal such as light
  • a detectable molecular spe ⁇ es such as a chromophore or fluorophore or electrochemically-reacQve molecule
  • This electromc method of electncal contact is based on electrons as earners of charge and elect ⁇ ty, as found in metals, conductors or semi-conductors. When an electrolyte is used to create elect ⁇ cal contact and allow elect ⁇ cal conduction, no direct electromc conduction is mvolved.
  • electrolyte When an electrolyte is used, the charge transferred during elect ⁇ cal conduction is earned by ionic spe ⁇ es (charged atoms and molecules) Thus, electromc conduction is not based on the movement of ionic spe ⁇ es as charge earners
  • An electrolyte is an lomc conductor capable of forming an elect ⁇ cal contact in all three states of matter gases, solids and liquids
  • electrolytes charged molecular spe ⁇ es move at the interface between an electrolyte and an electromc conductor or semi-conductor where electrochemistry can occur. This electrochemistry is dependent on the applied voltage, chemicals (Le. electrochemically-active compounds) in the electrolyte and the nature of the electromc conductor or semi-conductor surfaces.
  • Examples of materials which can be used m this invention to act as electrodes to carry out electrochemistry are without limitation: conducting or semi-conducting materials, composites, mixtures, alloys, blends or other such obvious de ⁇ vatives which combme many conducting, semi-conducting and non-conducting materials to produce materials which in the final form are able to act as conductors or semi-conductors.
  • Some examples are Si, GaAs, Pt, Ag, Au, C, Zn, Si, Ge, Sn, Pb, Hg, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Mo, Ru, Rh, Pd, Cd, In, Sb, W, Re, Ta, Os, Ir, Bi, Al, on oxide, indium/tin oxide (ITO), an ⁇ monv/nn oxide, electronically- conductive polymers such as polypyrole and polvthiophene Mate ⁇ als used as coatings mclude, carbon, gold, Pt, ITO and the conductive polymers
  • An example of a coated conductive material is Zelec ECP which is an electronicallv-conductive powder (DuPont, DE).
  • Zelec is available m two forms, one as a coating of antimony doped tin oxide on an inert core (silica, mica and titanium dioxide) or as solid particles of antimony doped tin oxide
  • an inert core silicon, mica and titanium dioxide
  • Au, Pt, ITO and Ag are the most favoured conducting mate ⁇ als of interest in the invention as they present a number of ideal properties including cost, stability, availability and expe ⁇ ence with these materials in a number of electrochemical systems. Also, these mate ⁇ als have already been used to manufacture numerous structures, forms and coatings.
  • C is of particular interest because of the number of different forms available and the number of differing electrochemical systems both in electrochemical applications and also in electrochemical assay devices which have made use of this matenal in its va ⁇ ous conducting or semi-conducting forms. Carbon is also readily de ⁇ vatized to saud numerous spe ⁇ ficallv modified surfaces such as in WO 97/33176 and WO 98/12539 which are hereby incorporated by reference in their entirety.
  • many other methods are known m the art for modifying carbon and other mate ⁇ als to render them useful for coupling Examples are cited in the following; US 5,667,667; P Allongue et al J Am Chem Soc 1997, 119, 201; C Sellim et al Material Science and Eng.1990, A126, (1990) 235-244; C Kozlowski et al J.
  • Examples of structures of conducting or semi-conducting mate ⁇ als suitable for the subject mvention are numerous. These structures take numerous shapes and forms, such as beads, particles, spheres, spheroids, cylinders, cones, plates, coatings, fibres, fibrils, nanotubes, nanoparticles, colloids, splinters, filings, springs, coils, amorphous particles, amorphous plates or coatings, and as a conductive coating take the form of two dimensional shapes of squares, triangles, rectangles, circles, pentagons, hexagons, stars etc.
  • the structures of most interest will be particles that are sphe ⁇ cal or spheres or similar, also fibres, fibrils and nanotubes.
  • Coatings take the form of conductive coatings on the surface of 3 dimensional structures, but can also be in the form of 2 dimensional shapes such as those produced by the evaporation of gold onto a surface which is masked to produce a desired shape on a surface.
  • Other examples of coating methods are without limitations, e.g. sputtering, screen printing, plating, polymensa ⁇ on, stamping and painting.
  • Many mate ⁇ als can be coated onto surfaces of flat or shaped structures thus producing a desired conducting or semi-conducting surface to form a structure for the purposes of the invention.
  • the potential exists to generate an array of such gold or carbon elements where each functions as separate bipolar electrodes as desc ⁇ bed for this mvention.
  • Other coating methods could also be used in this way to generate a senes of potential bipolar electrodes.
  • the present mvention using arrays of patterned conducting elements does not need to be in electromc contact with an outside circuit. As a result, the mvention solves a problem inherent to the apphcation of multiple electrodes or multiarray electrodes used in the development of electrochemical assay systems for multiple samples and multiple analyte detection systems.
  • particles or beads it is understood that particles or beads from cm to nm in size have been used in immunoassay applications ranging from tube-based assays to filter wicking assay systems. We anticipate the use of particles m these ranges as structures of the subject mvention.
  • the concentration of the conducting and/or semi-conducting matenal is such that the composite is itself conducting and/or semi-conducting.
  • the composites of this type are then equivalent to the conducting and/or semi-conducting mate ⁇ als of the subject mvention. These composites can then be used to form the structures of the mvention as desc ⁇ bed above forming for example fibres, particles, beads etc.
  • Other types of composite containing conducting and/or semi-conducting structures of the subject mvention are gels and sols and hydrogels.
  • the conducting material is dispersed or suspended in such a way that the conducting or semi-conducting structures do not render the composite electromcally conducting.
  • the amount of conducting or semi-conduc ⁇ ng structures is such that the final form of the composite is visible to vanous electromagnetic radiation and photon detection means.
  • such composites are amenable to the use of conventional electrochemical detection methods using electrochemical cells externally connected to a electromc circuit.
  • mate ⁇ als which are readily available and illustrate some of the forms contemplated in this mvention mclude: Aluminium powder, 20 micron; Beryllium powder, 10-20 micron; glassy carbon sphencal powder in va ⁇ ous sizes from 0.2 ⁇ m to 2,000 ⁇ m; graphite powders; graphite fibre 8 ⁇ m in diameter and 0.5 to 25 mm in length; gold powder/shot, sphencal, 2 ⁇ m to 3mm; gold flake, 2-15 ⁇ m; gold wires 0.05mm to 2mm dia; nickel powders, 2-7 micron, vanous forms; palladium powder, vanous forms from 0.25-7.5 micron; platinum wire 0.025mm to 2mm dia; platinum powder/ shot, va ⁇ ous forms, 0,2 ⁇ m to 3mm (Alfa Aesar, Ward Hill, MA); carbon nanotubes or fibrils (Materials and Electrochemical Research Corp, Arlington, AZ); long ordered carbon nanotubes from lO ⁇ m to 2mm have been desc ⁇ bed
  • the fab ⁇ ca ⁇ on of the structures of the subject mvention from the materials of the mvention is highly developed allowing a wide range of shapes and structures as outlined above.
  • so-called nanowires 55 ⁇ m long and about 250 nm in diameter have been made (Huber, CA, S ⁇ ence 1994, 263, p800).
  • nanowires of va ⁇ ous sizes and matenal be used advantageously in the present mvention as structures of the subject mvention.
  • structures of the subject mvention, as desc ⁇ bed above can be optionally coated to improve the electrochemistry or reduce interferences, for instance with gels, polymer layers or grafts and solid electrolytes layers which are known in the art. Detectable electrochemical reactions
  • Detectable electrochemical reactions consist of electrochemistry on a spe ⁇ es (a so-called electrochemically- detectable spe ⁇ es) which gives ⁇ se to a direcdy detectable spe ⁇ es such as photons of electromagnetic radiation or lndirecdy via production of a subsequendy-detectable spe ⁇ es or molecule such as a chemiluirunescent, fluorescent or coloured molecule or a molecule with an altered absorption spectrum, and electrochemically active spe ⁇ es
  • detectable electrochemical reactions exist producing electromagnetic radiation, electrochemically active molecules, chromogenic molecules and fluorogemc molecules.
  • the detectable reaction is direcdy coupled (i.e. comp ⁇ smg typically no more than four to five reaction steps) to the electrochemical reaction at the electrode surface but can also be lndirecdy coupled to the reactions at the electrode surface where numerous reactions linked to an initial electrode reaction result in the production of a detectable signal, spe ⁇ es and/or molecule.
  • the electrochemical reactions that produce electromagnetic radiation such as ECL, chem u ⁇ unescence, and electrolurmnescence, are easy to detect as they require simply a means to detect the electromagnetic radiation produced.
  • ECL and chemiluminescence the electromagnetic radiation is produced from electrochemical reactions ⁇ ther direcdy or by coupling to other electrochemically-produced reactants.
  • Va ⁇ ous forms of ECL are considered in this mvention, for example, ECL from Luminol with hydrogen peroxide, ECL from metal chelates and orgamc molecules which can occur in the presence of co-reactants such as oxalate (D Ege et al, 1984, Anal Chem 56, 2413-2417) and numerous amines as desc ⁇ bed in WO 97/33176 and EP 0441894B1 which are incorporated by reference in their entirety.
  • the ECL from Luminol with hydroperoxides e.g.
  • ECL is also generated from a number of metal chelates and orgamc molecules where use is made of the electrochemically-generated oxidised and reduced spe ⁇ es (generated at the anode(s) and cathode(s)) to produce the ex ⁇ ted state for light emission.
  • Light emission is also activated electrochemically from other chemilurmnescent molecules in addition to luminol such as acndinium ester and advantageously from stabilised dioxetanes such as those sold by Tropix (Waltham, MA),
  • stabilised dioxetanes these have typically been stabilised by the use of subs ⁇ tuent groups which can be removed enzymatically to destabihse the dioxetane and thus produce light
  • dioxetanes stabilised by groups which are or can be electrochemically removable provide for a wider range of potential chemist ⁇ es; alcohols, a ⁇ ds, and glycosides are contemplated as such groups (VG Mairanovsky, 1976, Angew.
  • fluorescence would be used as a detectable signal or label. Fluorescence could be used either via the synthesis or the activation of a fluorescent spe ⁇ es. For example, the local pH changes brought about in the proximity of a micro electrode can be sensed by the fluorescein activation caused bv the pH change in the local environment of the electrochemical reaction (BB Ratcliff et al 1996, Anal Chem 68, 2010-214).
  • Electrogenerated fluorescent spe ⁇ es are also detectable as demonstrated bv McLeod et al, (The Analyst, 1982, 107, 1-11)
  • the electrochemical reaction which is detectable, allows the detection of the conducting and/or semi-conducting structure.
  • the conducting and/or semi-conducting structure becomes the detectable spe ⁇ es and can be used to detect the analyte of interest. This property has been desc ⁇ bed for Al which can emit light on apphcation of a voltage at anodic and cathodic surfaces (Haapakka K et al, 1988, Anal Chun Acta 207, 195-210).
  • a conducting and/or semi-conducting structure coupled to a bmding spe ⁇ es to form the electrochemically- detectable spe ⁇ es is also contemplated. When bound or captured, this could subsequendy be detected using electrolytes with ECL and CL spe ⁇ es such as Ru(b ⁇ py), luminol.
  • the capture of a structure of the subject mvention would allow for considerable enhancement of the signal as a single bmding event would provide a bipolar electrode which could generate more light in an ECL reaction than from the capture of a few ECL active molecules.
  • Conducting and/or semi-conduc ⁇ ng structures made from or coated with electroluminescent matenal are contemplated. These coated structures of the subject mvention support the generation of hght from structures in the electrolyte when a suitable voltage is apphed without the need for additional chemistry.
  • the ECL labels of the subject mvention mclude numerous examples of both orgamc compounds and metal ion chelates.
  • Well known examples are t ⁇ s(2-2'-b ⁇ py ⁇ dine)ruthemum(ir) (Ru t ⁇ bipy), Ru t ⁇ s phen, Os t ⁇ bipy and numerous other metal chelates desc ⁇ bed m the hterature, and luminoL
  • Numerous examples are covered in the following publications: US5453356, US5591581, US5238808, US5221605, WO 97/33176, WO 96/28558 which are incorporated by reference in their entirety.
  • Al structures are known to luminesce when coupled to an electrochemical reaction m an electrolyte (Haapakka K et aL 1988, Anal Chun Acta 207, 195-210).
  • Examples of other compounds that produce ECL are 9,10-d ⁇ phenylanthracene, rubrene, b ⁇ s(tn-n- hexyls ⁇ loxy)(2,3- ⁇ hthalocyamnato)s ⁇ hcon and its dimer, b ⁇ s(t ⁇ -n-hexvlsdoxy)(2 ⁇ -naphtalocyamnato)s ⁇ hcon > t ⁇ s(4-vmyl-4'-methyl-2 ⁇ 'b ⁇ py ⁇ dyI)n ⁇ themum(II) and its polymers, lu ⁇ genin, ac ⁇ dimum esters, rhodamine 6G, europium chelates, cryptates and mixed-hgand chelate/cryptand complexes (
  • the electrolyte of the present mvention typically contams ionic spe ⁇ es in liquids, solids and gases to allow elect ⁇ cal conducting A charge passing through an electrolyte is transferred via movement of the lomc spe ⁇ es m the bulk liquid, solid or gas.
  • the ionic spe ⁇ es is also mvolved in the control of the ionic strength and/or the pH of the electrolyte such that it is optimal for the assay of the analyte of interest and/or the electrochemistry.
  • An example of an electrolyte that can be used for ECL with Ru is phosphate buffer used as electrolyte and pH control, combmed with tnpropylamine as an amine coreactant to support the ECL electrochemistry.
  • buffers for example borate, phosphate, t ⁇ s, HEPES, MES, MOPS, PIPES, acetate, t ⁇ ethylarrune, tnpropylamine, bicarbonate with the approp ⁇ ate counter ion
  • the buffer would be a coreactant, such as amine-containing buffers that can act as coreactants in Ru (2,2- b ⁇ py ⁇ dyl)3 2+ -based ECL.
  • the ionic strength of the medium can be controlled typically by adding salts, or be established by controlling the concentrations of the buffer and/or the coreactants. Examples of salts which are used to control the ionic strength are numerous.
  • Examples m include salts of K, Na, Ca, ammonium, guanidinium.
  • electrolytes of the subject mvention are based on water solutions and orgamc solvents.
  • Advantageously electrolytes of the subject mvention are based on water solutions.
  • Orgamc solvents in some cases are advantageous during the electrochemical reaction. It will be understood that mixed solvents are also used.
  • formamide is used advantageously in DNA probe applications.
  • components are also added to improve assay conditions.
  • Typical examples of components used to improve lmmunoassays include detergents, other proteins and hpid components. These other components are known m the art and are typically optimised for va ⁇ ous assays under the va ⁇ ous conditions of differing assay systems. The optimisation of these components is well known in the art and forms an integral part of assay optimisations. The use of other components to enhance vanous aspects of nucl ⁇ c a ⁇ d probes is also known in the an. Instruments Basically, the instruments integrate a luminometer, a voltage control ⁇ r ⁇ nt and a cell with two externally electromcally connected electrodes.
  • the apparatus includes a pump or other fluid-handling components and a flow-through cell with externally electromcally conne ⁇ ed electrodes.
  • the instruments of the subject invention advantageously have a cell which contams at least one electrolyte. This cell contams at least two electrodes which are m electromc contact with an external elect ⁇ cal circuit which is able to apply a voltage. These two electrodes are in contact within the cell via the electrolyte within the cell. It is possible to design cells of va ⁇ ous shapes and configurations such as flow-through cells, single- chamber cells which allow the introduction of electrolyte at desired times.
  • the cell and electrodes are a single module which can be inserted into the instrument to make contact
  • Externally electromcally connected electrodes isolated from a central zone or at least from each other by a membrane, gel or other material which is semi permeable to ions and thus maintains contact between the two externally electromcally connected electrodes via the electrolyte are also contemplated.
  • This use of a semi permeable membrane isolates the sample from the externally electromcally connected electrodes and from the electrolyte in which they are immersed.
  • This method of isolating the externally electromcally connected electrodes prevents both the detectors and the sample from bemg affected by the electrochemistry occurring on the externally electronically connected electrodes
  • va ⁇ ous instruments contemplated would be controlled by a microprocessor ⁇ ther direcdy by incorporation of a microprocessor into the instrument and/or by an external microprocessor such as a computer.
  • Software for controlling such mterfaces is well known to those in the art for custom applications to make use of internal and external microprocessor control e.g. Labview from National Instruments (Austin Tx) www.nattnst.com.
  • simple electromc circuits can be constructed to control the apphcation of the voltage to the cell.
  • an instrument of the sub j ect invention contams a cell which is able to contain an electrolyte of interest for the detection and/or quantitation of an analyte(s) of interest which further consists of three zones or functionally distinct areas. Two of the zones flank a central zone and contain externally (to the cell) electromcally connected electrodes. These two externally electromcally connected electrodes are used to apply voltages to the central zone of the cell. The two externally electtonicallv connected electrodes are externally connected to a controlled voltage source.
  • this controlled voltage source is under the control of a computer or is controlled by other means such as a simple electncal circuit or mechanical means for triggering the apphcation of the voltage.
  • the controlled voltage apphed to at least the central zone of the cell is initiated by an operator or by determining the presence of, or placing, the sample electrolytes in the central zone of the cell.
  • the sample electrolyte contams the conducting and/or semi-conducting structures of the subject mvention along with the compound(s) which are able electrochemically to produce a detectable spe ⁇ es allowing detection of analytes.
  • the two externally electromcally connected electrodes electromcally connected to the controlled voltage source are thus able to apply a desired voltage gradient to the central zone of the cell controlled by electromc means including a simple electromc circuit and/or microprocessor.
  • electromc means including a simple electromc circuit and/or microprocessor.
  • the apphcation of this desired voltage to the central zone of the cell allows the conducting and/or semi-conducting structures of the subject mvention to carry out electrochemistry.
  • This desired electrochemistry results in the formation direcdy or lndirecdy of a detectable spe ⁇ es which when detected allows the detection and/or quantitation of the analyte of interest
  • the central zone of the cell is fashioned in such a way that it readily allows the detection of the detectable spe ⁇ es generated by the electrochemistry.
  • the central zone will contain at least one window.
  • This window will permit the detection of electromagnetic radiation from the central zone.
  • the window is present and additional externally and electromcally connected electrodes are introduced through the open window into the central zone, or into a zone in a flow-path from the central zone, to allow electrochemical detection of the detectable spe ⁇ es using conventional and understood electrode configurations in combmation with conventional electrochemical methods such as amperometnc or potentiomet ⁇ c methods. Examples of these are glucose and oxygen sensors and oxygen electrodes.
  • the window will permit photon detection by a means of choice to detect and/or quan ⁇ tate the photons from the central zone.
  • the central zone would allow a beam of photons to pass through the sample in the central zone and be analysed and/or detected and/or quanntated The analysis of this beam of photons after passing through the sample in the central zone would determine whether the intensity of the photon beam had changed and/or if the spectral properties of the photon beam had been changed.
  • the beam of photons would have a wavelength selected to interact optimally with the detectable chromogenic spe ⁇ es of interest.
  • the central zone would allow a beam of photons to pass through the sample and the photons emitted from fluorescent molecules present in the sample to be analysed and/or detected and/or quanntated.
  • the analysis of these photons emitted from fluorescent molecules, in the central zone would determine whether the spectrum had changed with respect to the ln ⁇ dent beam of photons.
  • the beam of photons would have a wavelength selected to interact optimally with the detectable fluorogemc spe ⁇ es of interest and the detector would be optimised to detect and/or quan ⁇ tate the emitted photons from the detectable fluorogemc spe ⁇ es.
  • time- resolved fluorescence is also a contemplated detection means.
  • fluorescence and/or other energy transfers is also contemplated as a means of signal enhancement and discrimination, as used in other assay systems known in the art
  • Other detection means are also contemplated by this assay system and instrument, such as electrochemical means for the products of the electrochemical reactions at the surface of the structures of the subject mvention. Cells of the instrument
  • Va ⁇ ous cells for the instrument of the subject mvention are contemplated and it is understood that numerous potential examples are possible. Examples are desc ⁇ bed in detail m the section "Examples" In the case for an instrument able to analyse clinical samples, a flow-through cell with the three zones descnbed above would be advantageous. When usmg the subject mvention with rapid immunochromatography-based assays, a cell would be a section of such a device. This section could be removable and is potentially a disposable element.
  • the structures of the mvention could be coated onto the surface of the well in a microtitre plate, typically on the bottom or portions of this part of the well, either as a single contiguous conducting or semi-conducting structure or advantageously as multiple elements in the size range of 1 ⁇ m to 1 mm as measured between the edges of the mdividual structures m the plane between the externally electromcally connected electrodes.
  • the wells in this multiwell format could have the externally electromcally connected electrodes within the well or these could be introduced at a suitable time to apply the electncal field needed to render the conducting or semi-conducting structures bipolar thus stimulating electrochemistry This would result in the production of a detectable signal
  • Multiwell plates constructed using composite mate ⁇ als containing conducting and/or semi-conducnng structures of the subject invention is also contemplated. Multiwell plates so constructed would possess well surfaces of conducting and/or semiconducting structures able to act as bipolar electrodes in an apphed field.
  • the conducting or semi-conducting structures of the subject mvention could take the form of multiple mdividual patterns or shapes on the surface of a non-conducting matenal forming a multitude of potential assay sites rendered bipolar in an apphed elect ⁇ cal field. These bipolar structures of the subject invention would then be capable of electrochemistry m electrolytes known to the art.
  • the surface with its multiple mdividual patterns or shapes of conducting or semi-conducting matenal form the bottom of a well in an assay plate with many other such surfaces as found in a microtitre plate or are part of a flow-through system as a means for introduction of sample and/or reagents.
  • the surface with its multiple structures must be in contact with an electrolyte and must have at least one window to allow the detection of the detectable spe ⁇ es. This provides for significant advantages over the current art which requires external electromc connections to each element. Further examples of cells are descnbed in the section "Assay formats" and the section "Examples”.
  • a voltage is apphed to the sample.
  • the following section desc ⁇ bes some examples of these and gives examples of the apphed voltage waveforms.
  • Apphed voltage profiles can be those seen in typical electrochemical studies and cells with the exception that these are not limited to the low voltages seen in conventional electrochemical cells (less than 5 volts).
  • the voltages apphed to the structures of the subject mvention range from 2 volts/cm to 50,000 volts/cm or the limit of the solvent/electrolyte used.
  • voltages range from 20 volts/cm to 50,000 volts/cm or the limit of the solvent/electrolyte used. More advantageously, the voltages range from 50 volts/cm to 50,000 volts/cm or the limit of the solvent/electrolyte used.
  • the voltages range from
  • the voltage apphed to the cell of the subject mvention via the externally and electromcally connected electrodes can take multiple forms.
  • voltage apphed in a step form where the potential is raised to a given value and held for a time and then removed is also contemplated. It is also contemplated that this type of step is apphed repeatedly to give a square wave form with a wide range of frequen ⁇ es from 0.001-10,000 cycles per sec, advantageously this is from 0.001 to 1,000 cycles per sec, more advantageously this is from 0.01 to 1,000 cycles per sec.
  • Voltage scans or waveforms containing portions of reverse potentials are also contemplated.
  • the step potential would be desc ⁇ bed as having a negative and then a positive portion and this single unit repeated to give an alternating square wave form.
  • the voltage apphed in such a way that it follows a ramp up and down in voltage with or without negative or reverse pola ⁇ ty portions to the ramp is also contemplated.
  • These ramps constructed around numerous potential waveforms such as sin waves, saw tooth waves, multiple component step waves and combinations of these are also contemplated.
  • advantageously having at least a lower voltage portion in such wave forms p ⁇ or to the ex ⁇ ta ⁇ on of the electrochemically detectable spe ⁇ es is contemplated.
  • cycles take from 0.01 to 1600 sees to complete, advantageously this is from 0.1 to 600 seconds, more advantageously this is from 1 to 300 seconds, most advantageously this is from 1 to 60 seconds.
  • the measuring cycle in the instrument consists of the introduction of the sample into the central zone of the cell either by flow (in a flow-through system), by robot arm or other manual or mechanical pipetting means, or by pla ⁇ ng a cartridge and/or disposable element containing the electrodes, conducting and/or semiconducting structures and sample into the instrument
  • the sample is then subjected to a voltage waveform or pulse as outlined above and the detectable electrochemical reaction is then detected
  • Sub j ecting the sample to somes or ultrasonics to improve the kinetics of the bmding reaction and/or the electrochemistry is contemplated and has been desc ⁇ bed previously for these reactions and is known in the art (K Suslick, S ⁇ ence, 1990, 247, 1439-1445 and D Walton et al mectrochimica Acta, 1993, 38, 307-310)
  • Mixing achieved via the use of magnetic methods or other mechanical methods known m the art which are known to achieve significant fluid mixing is also contemplated These vanous forms of mixing are advantageous
  • the voltages and waveforms descnbed above are apphed du ⁇ ng mixing or sonication or ultrasomcation.
  • multiple (advantageously from 2 to 20) externally and electromcally connected electrodes are used m a cell to achieve the apphcation of a voltage field at va ⁇ ous angles with or without the apphcation of alternating positive and negative fields to achieve activation of a greater surface of the structures of the subject mvention
  • externally and electromcally connected electrodes could be positioned at the eight positions of a sphere, or comers of a cube or rectangle defining the central zone of the cell These electrodes could then be used in pairs or other va ⁇ ous combinations to apply both negative and positive voltages to the cell
  • These voltages could be controlled bv a computer or mechanical device to apply a sequence of voltages to the different electrodes in such a way that all faces of the conducting and/or semi-conducnng matenal are activated to carry out the desired electrochemistry to
  • va ⁇ ous faces of the conducting and/or semi-conducting structures of the subject mvention could also be used in combination with multiple detectors of electromagnetic radiation to permit correlation of apphed voltages and electromagnetic radiation from va ⁇ ous faces to be made thus allowing a more effective determination and elimination of background signals in the system and from the detectors and their associated circuits
  • the va ⁇ ous forms in which the electrochemicallv-detectable signal can be detected are numerous and are known in the art, for example in the case of ECL and chemiluminescence, a light detection method based on the use of photomul ⁇ plier tubes and photodiodes (luminometers), CCD cameras, film and by eye, in the case of a fluorescent molecule generated bv the electrochemistry, a detector based on a fluonmeter would be used; m the case of a chromogenic or other molecule with a new absorption spectrum, a spectrophotometer system would be used, m the case of an electrochemically active molecule generated as the electrochemically detectable
  • Va ⁇ ous assays are desc ⁇ bed for the subject mvention and these can be earned out making use of numerous bmding species
  • a molecular spe ⁇ es mcludes chemicals from single spe ⁇ es atoms or ions (I e Ca + ) to complex multiple atom compounds (I e antibodies)
  • Most binding spe ⁇ es are defined by the existence of a partner binding species since, to be a binding spe ⁇ es, the molecular spe ⁇ es must bmd to another molecular spe ⁇ es, which mcludes the same molecular spe ⁇ es.
  • an antibody developed such that it will bmd to itself. In many cases, this type of self bmding is not valuable but can be useful in certain cases, for example, where development of signal amplification is of interest.
  • these two spe ⁇ es can be considered to be bmdmg spe ⁇ es since they are both able to bmd to another molecular spe ⁇ es.
  • the antibody bmding spe ⁇ es' partner is the antigen and the antigen bmding spe ⁇ es' partner is the antibody.
  • the antibody and the antigen can both be considered to be bmdmg spe ⁇ es.
  • the following is a partial list of such bmding spe ⁇ es contemplated in the subject mvention: antibodies, antigens, receptors, enzymes, enzyme inhibitors, drugs, hormones, gands, receptor hgands, lectins, selectins, mtegnns, RNA, DNA, nucleic a ⁇ d analogues, nucl ⁇ c a ⁇ d bmding protons, chelating hgands.
  • These bmding spe ⁇ es are all contemplated as analytes that can be detected and/or quanntated by the subject mvention.
  • Analytes of the subje ⁇ mvention are numerous, including the binding spe ⁇ es descnbed above, but also mclude molecules which are not bmdmg spe ⁇ es or can be detected without being part of a binding-spe ⁇ es to binding-spe ⁇ es lntera ⁇ ion.
  • the character which defines these groups is their ability to interact with the electrochemistry in a detectable manner, this interaction occurring because of the apphed voltage on the structures of conducting and/or semi-conducting materials not m external electromc contact.
  • Also considered as analytes are molecules able direcdy or lndirecdy to produce, destroy or metabolise electrochemically detectable spe ⁇ es.
  • An example would be the detection and/or quantitation of amine- containing compounds which act as a coreactant in the generation of ECL from va ⁇ ous metal chelates such as Ru(bpy) 3 2+ Examples of this are contained m EP 0 441 894 Bl which is incorporated by reference in its entirety. Other examples are tnpropylamine, NAD(P)H, and hydrolysed beta lactams.
  • ECL labels (as desc ⁇ bed earlier) and ECL active spe ⁇ es such as Ru(bpy)3 2' , ruberene, luminol, and dioxetanes are also considered as analytes No bmd g interactions occur in this reaction
  • the product of this reaction (hydrogen peroxide) acts as a coreactant with luminol to electrochemicallv generate hght. It would be understood from such a reaction that in addition to glucose as an analyte, glucose oxidase and hydrogen peroxide could also be analytes.
  • an analyte subjected to a se ⁇ es of reactions giving ⁇ se to a molecular spe ⁇ es which can direcdy parti ⁇ pate in modifying (enhan ⁇ ng or inhibiting) a detectable electrochemical reaction is contemplated.
  • An example of this is seen in the case of a glucose assay based on a well understood assav for glucose which first kinases the glucose (with ATP and the enzyme hexose kinase) to form glucose-6-phosphate.
  • analytes mclude enzyme substrates, enzyme products, enzymes, whole cells, viruses, sub cellular particles, nucl ⁇ c a ⁇ ds, polysaccha ⁇ des, proteins, glycoproteins, lipoproteins, hpopolysaccha ⁇ des, pids, fatty a ⁇ ds, peptides, cellular metabolites, hormones, pharmacological agents, tranquillisers, steroids, vitamins, ammo a ⁇ ds, sugars, and non biological polymers Assay formats
  • the subject invention allows for electrochemical stimulation withm a sample volume, generating a detectable signal which allows for the detection and/or quantitation of analytes within the sample volume and to a greater extent than previously possible because of the effective extension of the electrode surface area available.
  • This ability to generate a detectable signal by stimulation of electrochemistry withm the volume of a sample provides the following advantages;
  • spatial discrimination allows multiple samples to be analysed and provides the potential for photographic- ke resolution withm a sample l e. a gel
  • This spatial discrimination can also be three dimensional.
  • Ohgonudeotide p ⁇ mers are labelled with an electrochemiluirunescent (ECL) label ORIGEN TAG (IGEN, Gaithersburg MD) following methods desc ⁇ bed by the manufacturer (apphcation notes descnbing PCR are available on the internet at http:/ / ww.igen.com). These PCR p ⁇ mers are then used in a PCR to amplify and generate an ECL-labelled PCR produ ⁇ which is dependent on the presence of DNA complementary to the labelled PCR p ⁇ mers.
  • ECL electrochemiluirunescent
  • the PCR product can now be run on a standard agarose or polvacrylamide gel containing for example advantageously carbon nanotubes or carbon fibres (Alfa Aesar; Mate ⁇ als and Electrochemical Research Corp, Arlington, AZ, Pan ZW et al, Nature 1998, 394, p631 and Fan S et al, 1999, S ⁇ ence, 283, p512) as conducting and/or semi-conducting material.
  • carbon nanotubes or carbon fibres Alfa Aesar; Mate ⁇ als and Electrochemical Research Corp, Arlington, AZ, Pan ZW et al, Nature 1998, 394, p631 and Fan S et al, 1999, S ⁇ ence, 283, p512
  • These nanotubes would be included at such a concentration that the gel is not conductive and is not totally opaque such that no ECL would be visible, advantageously 0.1-10 ⁇ g/mL It is understood that other suitable conducting or semi-conducting mate ⁇ als (as desc ⁇ bed above) are also used
  • the gel is then loaded with the PCR products and the DNA subjected to electrophoresis at voltage gradients typical for the separation of PCR products ⁇ .e. less than lOOv/cm. After the electrophoretic separation, the gel is then soaked in ORIGEN assav buffer (IGEN, Gaithersburg, MD) for 30-60 mm with three changes of buffer.
  • ORIGEN assav buffer IGEN, Gaithersburg, MD
  • This gel is then placed m a high voltage chamber contained withm a black box to exclude as much hght as possible with electrolyte in contact at each end of the gel as in a typical electrophoresis chamber
  • the high voltage container has an electrode at each end as is typical of an electrophoresis chamber with the gel between these electrodes
  • the gel is visualised by a photomultipher tube, film or CCD camera
  • the door to the black box containing the gel, high voltage chamber and photon detection system (PMT(s) or CCD camera or photod ⁇ ode(s)) is closed to create a substantially light-free environment
  • the high voltage chamber is then subjected to a high voltage which typically mcreases at a rate of 100 v/ s to 10 kv/s but could also be in the range of lOv/s to 1,000 kv/s.
  • This voltage ramp is apphed for 0 1 sec or longer. Du ⁇ ng the apphcation of these high voltages, peak voltages are controlled to prevent arcing which would indicate an excessive voltage. The voltages reached are advantageously from 100 v/cm to 10,000 v/ cm. Du ⁇ ng the high voltage sweep the light produced is detected by the CCD camera (or other hght detection means) and the location and/or amount of the PCR product is determined
  • the electrophoresis buffer would also be the ECL buffer as in the case of a buffer (as desc ⁇ bed earlier) which contams amines useful for the generation of ECL, for example t ⁇ propylamine-phosphate, tnpropylamine-acetate, tnethvlamine-borate, t ⁇ propylamme-borate, PIPES and HEPES (WO 97/33176).
  • ECL buffers are ProCell (Boeh ⁇ nger Mannheim) and ORIGEN assay buffer (IGEN).
  • ECL electrophoresis
  • tns(2,2-b ⁇ pyndyl) ruthenium oxalate system for example, the gel could be pulsed to the higher voltages at desired times du ⁇ ng the lower voltages used for the electrophoretic separations to activate the ECL labels on the labelled molecules.
  • the higher voltages are required to render the carbon nanotubes or fibrils bipolar (generating an anodic and cathodic face on at least some of the carbon nanotubes or fibrils, or other structures of conducting and/or semi-conducting matenal) resulting in the activation of the ECL label contained in the PCR products separated on the gel, as descnbed above, or other labelled molecules
  • This high voltage pulse used to render the carbon nanotubes or fib ⁇ ls bipolar and activate the electrochemistry on part of the surface of at least some of the carbon nanotubes or fib ⁇ ls could thus be part of the electrophoresis protocol. This would allow the electrophoretic migration to be followed over time by mtermittent activation of the ECL labels attached to the labelled PCR products.
  • a further modification of the above example concerning the in situ detection of an analyte of mterest withm gels or other separation media is also contemplated.
  • This method contemplates the detection of numerous molecules able to support ECL as has been demonstrated for numerous amine-containing molecules such as TPA, TEA, PIPES, HEPES, beta-lactams, NADH, and enzymes coupled to XAD or NADP etc.
  • This luminol ECL can also be coupled to the detection of hydroperoxides such as pid hydroperoxides
  • the conducting or semi-conducnng matenal is precoated with an ECL active label sensitive to the presence of amines or other potential coreactants that enhance ECL, e.g. Ru (2,2-b ⁇ py ⁇ dyl)3 2+ when these amines or other potential co-reactants are in close proximity to the dispersed conducting or semi-conduc ⁇ ng matenal coated with the ECL active spe ⁇ es.
  • This type of detector system would be useful in the detection of mate ⁇ als in gels, matenal elu ⁇ ng from columns du ⁇ ng pu ⁇ fication, du ⁇ ng capillary electrophoresis and obviate the problems with the application of conventional electrochemical stimulation which requires sample withm a narrow region of an externally electromcally connected electrode surface which, bemg able to analyse or activate only a verv small portion of any given sample, results in poor sensitivity for these conventional methods.
  • the coated conducting or semiconducting matenal would be ex ⁇ ted by the apphcation of an external voltage to generate bipolar faces on at least some of the dispersed conducting or semi-conducting matenal, and thereby allow the detection of the ECL co-reactants such as amines, beta-lactams, NADH, NADPH and peroxides.
  • the ECL acnve spe ⁇ es are added m solution i.e. in the gel, HPLC or capillary electrophoresis buffers. This would remove the need to immobilise the ECL spe ⁇ es to the conducting and/or semiconducting structures of the subject mvention.
  • this system could be used to detect ECL labels direcdy or by supporting ECL with a co-reactant.
  • peroxides could be detected e.g. hydrogen peroxide or lipid hydroperoxides.
  • these conducting or semi-conducting materials coated with an ECL active spe ⁇ es sensitive to co-reactants such as amines, peroxides, beta-lactams, NADH and NADPH, could be used as suspended electrode systems for the detection of va ⁇ ous molecules which affect the concentration of these co-reactants.
  • enzymes acting on va ⁇ ous substrates can affect the concentration of these co-reactants allowing detection of ⁇ ther the enzyme or the substrates of the enzyme.
  • An example would be dehydrogenases that generate NADH or NADPH, e.g. glucose-6-phosphate dehydrogenase which can be used in a glucose assay when coupled to hexokinase as desc ⁇ bed earlier.
  • the analyte of mterest is a molecule which can be spe ⁇ fi ⁇ dly acted on by a dehydrogenase to produce NADH or ADPH. This is then detected and quanntated by the activation of ECL. In the previous example this is glucose-6-phosphate.
  • the assay could be earned out as follows: the sample would be mixed with the appropriate dehydrogenase and the appropriate co-factor such as NAD or NADP.
  • This enzyme/substrate/co-fa ⁇ or mix would then be supplemented with particles, fibres or fib ⁇ ls of conducting or semi-conducting material precoated with an ECL active label sensitive to the presence of NADH or NADPH, e.g. Ru (2,2-b ⁇ pv ⁇ dyl) 3 2+ .
  • the conducting or semi-conducnng material could be added with an ECL acnve spe ⁇ es such as Ru (2,2-b ⁇ py ⁇ dyl) 3 2+ free m solution.
  • the detection of antigens, antibodies, receptors, hgands and nucl ⁇ c a ⁇ ds and their analogues is possible.
  • This embodiment relates to an immunoassay, nucl ⁇ c a ⁇ d assay or other similar bmdmg assays earned out usmg a filter st ⁇ p or wicking method and also called immunochromatographic tests or lateral-flow tests. Examples of these are found in US 5591645, US 4861711, EP 0 258 963 A2, US 4703017 and US 5008080 which are hereby incorporated by reference in its entirety.
  • This embodiment relates to the electrochemical activation of at least part of such a filter or flow- supporting matenal such that an analyte contamed withm or bound throughout the body of the filter or flow- supporting matenal is dete ⁇ ed and/or quanntated.
  • These tests usmg a filter snip or wicking method and also called immunochromatographic tests or lateral-flow tests, offer the beauty of simph ⁇ ty. They involve simply the apphcation of a sample (e.g. blood, u ⁇ ne, sahva).
  • the analyte-labelled antibody complex interacts with the immobilised capture antibody it is captured by bmding to an available epitope on the analyte of mterest, resulting m the immobilisation of the label which is attached to the labelled antibody and complexed with the analyte.
  • the fluid continues to wick or flow past the immobilised anubody carrying any un-complexed (not bound to analvte of mterest) labelled antibody awav from the immobilised capture antibody.
  • an ECL label could be bound to the antibody (IGEN, Inc, Gaithersburg, MD) although other labels are possible, and the filter matrix or supporting phase could be manufactured to mclude structures of conducting and/or semi-conducting mate ⁇ als. Examples of such conducting and/or se ⁇ u-conduc ⁇ ng mate ⁇ als mclude, carbon nanotubes, carbon fibres, glassv carbon particles, Au and/or Pt nanowires.
  • These conducting materials would be mixed with the mate ⁇ als used in the manufacture of the filter membranes or fibre wicking matenal m such a way that they would be immobilised within the bulk of the membrane or supporting phase m such a way that the supporting phase or membrane is not transformed mto an electromc conducting or semi-conducting matenal.
  • An alternative embodiment would be to immobilise the capture antibody onto a conducting or semiconducting matenal fi e.
  • the conducting particles or fibres could be chemically or physically trapped in the filter or flow-supporting material by impregnation of the conducting or semi-conducting particles or fibres usmg printing or injection methods followed by antibody immobilisation either onto or withm close proximity of the conducting or semiconducting matenal
  • Potentially appropriate conducting and semi-conducting mate ⁇ als are carbon nanotubes, carbon fibres, C, Au and/or Pt nanowires
  • the ECL hght is detected via a number of understood methods for light detection and/or quantitation, including eye, photography, photodiode, photomultipher or photocell. It will be understood by those skilled in the art that this method of analyte detection usmg a bmdmg reaction between antibody and analyte could also be apphed without a labelled antibody but via the use of a labelled analyte which competes with the sample analyte for bmdmg to the capture antibody.
  • these immunoassay formats are apphcable to numerous bmd g assays based on other well understood binding interactions, e g nucleic a ⁇ d - nucl ⁇ c a ⁇ d, receptor- gand, nucleic a ⁇ d analogues-nucleic a ⁇ d. and nucleic acid-protein It is also understood that these immunochromatographic assays include those developed with liquid reagents not d ⁇ ed onto the immunochromatographic assay device
  • the assay is earned out without the need for a so-called wash step to remove the unbound or free label.
  • the bmdmg of the labelled species modulated in the presence of the analvte is detected due to the effective concentration of the label on the surface of the conducting or semi-conducting matenal which allows selective EC activation of the bound labelled spe ⁇ es over that of the unbound
  • This method - which allows the assays to be earned out without a wash - is of great benefit in that it allows for rapid testing and simplification of the diagnostic system device
  • a filter st ⁇ p or wicking method also called immunochromatographic tests or lateral-flow tests
  • the structures of conducting or semi-conducting material - advantageously with the longest dimension bemg less then 200um - would be the label and a binding spe ⁇ es attached to this label.
  • the assav would be conducted in the normal manner for such immunochromatographic tests or lateral-flow tests, resulting in the capture of the structures of conducting or semi-conducting material on the capture antibody at a predetermined site in the immunochromatographic tests or lateral-flow tests st ⁇ p or cartridge based on the presence of analyte.
  • Such assays already exist and make use of latex beads, colloidal gold or silver as a label on the antibody (Boeh ⁇ nger Mannheim) but these use visual detection of the particles and no use is made of the electrochemical properties of the conducting or semi-conducting matenal.
  • Stabihsed dioxetanes are also considered since they can be activated by electrochemical methods. Visible signals can also be generated e.g. bv electropolyme ⁇ sation to generate polypyrole and polvthiophenes
  • Conducting or semi-conducting material used as the label can be electroluminescent or chemilum escent with or without addition of active spe ⁇ es to the buffer used for the assay.
  • An example of such an electroluminescent or chemiluminescent material, active without addition of active spe ⁇ es is Al (Haapakka K et al, 1988, Anal Chim Acta 207, 195-210). It is also contemplated that other electroluminescent compounds be used in this way.
  • This mode of detection for the conducting or semi-conducting label is supe ⁇ or to known visual methods as the labels can be detected before they are visible or they can be amplified to become visible as in the case of electropolvmensation
  • the mvention improves on the current immunochromatographic tests or lateral-flow tests which use conducting or semi-conducnng materials as labels
  • these filter wicking assays are also apphed to the detection of analytes without the need for a binding spe ⁇ es.
  • ECL labels or co-reactants are detected.
  • Examples of potentially-detectable analvtes have already be desc ⁇ bed above for the subject mvention.
  • a good example would be the detection of glucose, where the apphcation of the sample to the filter-wicking substrate b ⁇ ngs the glucose mto contact with glucose oxidase and luminol.
  • the glucose m the sample is acted upon bv the glucose oxidase to generate hvdrogen peroxide and this wicks up the wicking substrate and comes mto contact with the structures of conducting and/or semi-conducting matenal of the subject invention.
  • the wicking substrate with the sample is then subject to an apphed external voltage which activates the structures of conducting and/or semi-conducting material to generate ECL from the luminol and the hydrogen peroxide produced bv the action of glucose oxidase on the glucose in the sample
  • This ECL is used to detect and/or quantitate the glucose in the sample
  • the value of a glucose assay in this format is that it allows the separation of the sample pnor to the enzyme reaction and detection of the ECL This is most valuable as, for example in the case of a blood sample, it allows the removal of the red and white blood cells and other interfering substances
  • a glucose assay earned out with dehydrogenases usmg the ECL from the Ru(bpy)3 2+ and NAD(P)H reaction based on the coupled enzyme reaction employed for glucose analysis of hexokinase and glucose-6-phosphate dehydrogenase (as descnbed above in analytes) is also an example of a filter wicking
  • filter wicking assays are thus not rest ⁇ cted to the detection of bmdmg reactions but can be used to detect enzymes, substrates of enzymes, ECL co-reactants, ECL labels, ECL active molecules and EC reactive spe ⁇ es as desc ⁇ bed earlier for the subject mvention.
  • an advantageous embodiment of the present mvention is a bmdmg assay such as an immunoassay, receptor, gand or nucl ⁇ c a ⁇ d assay where small conducting or semi-conducting structures are coated with one of the bmdmg spe ⁇ es to form the ⁇ ipture phase of the assay.
  • bmdmg assay such as an immunoassay, receptor, gand or nucl ⁇ c a ⁇ d assay where small conducting or semi-conducting structures are coated with one of the bmdmg spe ⁇ es to form the ⁇ ipture phase of the assay.
  • glassv carbon (or graphiuc) structures are activated by standard methods to generate, for example, NHS ester-activated structures (L.
  • any non specific bmdmg in the assay is blocked by incubation with a non spe ⁇ fic proton such as BSA and/or an amino-containing compound such as erhanolamine which react with any remaining NHS groups and bmd to non-spe ⁇ fic bmdmg sites on the surface of the structures
  • a spe ⁇ fic bmdmg spe ⁇ es such as an antibody (or streptavidin) and blocked are now ready to be used as the sohd phase in a bmdmg assay
  • these particles are added to a mixture of sample and another bmdmg spe ⁇ es which is labelled with an EC ac ⁇ vatable label and able to bind to the analyte of mterest This mixture is allowed to incubate typically for 1-60 mm During this time, the labelled bmdmg spe ⁇ es, for example an antibody, binds to the analyte of mterest and this
  • the sample is now direcdy activated by the apphcation of a voltage such that bipolar faces are generated on at least part of at least some of the glassy carbon structures
  • the bipolar faces so generated are of suffi ⁇ it voltage that thev are able to activate the EC activatable label(s)
  • the EC acovatable label is an ECL label which is detected usmg standard methods for the detection of hght, e g PMT, photodiode, film, eve, CCD etc
  • the glassy carbon structures are first washed with a suitable buffer pnor to the activation of the EC activatable labels as descnbed above.
  • the washing of the glassy carbon structures could be achieved in vanous ways Usmg magnetic structures as the basis for the assay would allow for a method based on magnetic capture, but centnfugation and filtration are potentially valuable methods which could be apphed.
  • the structures could be readily collected and washed on the surface of a filter which could form the matrix on which the structures could be activated by the apphcation of a voltage across the filter.
  • Contemplated is the apphcation of a voltage across a filter making use of any axis and wherein the axis of apphed voltage is also optionally changed, rotated or scanned and the pola ⁇ tv of the field changed such that more of the captured structures and /or more of the structures coated with immobihsed EC activatable labels are activated.
  • an alternating voltage is apphed with va ⁇ ous frequen ⁇ es and waveforms (or pulses).
  • the apphcation of somes used to improve signal generation, bmdmg kinetics i.e.
  • the structures of conducting and/or semi-conducnng matenal havmg a magnenc component allowing capture for washing and/or optimal positioning of the structures with respect to the voltage field and/or detector are contemplated.
  • An alternative embodiment of the assay descnbed above for glassy (and graphitic) carbon structures is an assay that detects an analvte which is in competition with the structure-bound bmdmg spe ⁇ es for bmding with a labelled equivalent of the analvte.
  • the label in this example is the same as desc ⁇ bed above, bemg an EC activatable label which results in the production of a detectable signal or EC detectable spe ⁇ es.
  • the- sample typically will have added to it the labelled equivalent of the analvte followed bv the addition of the structures or other conducting or semi-conducting matenal coated with a bmdmg spe ⁇ es spe ⁇ fic for the analyte of mterest or capable of bmding to a bmdmg spe ⁇ es spe ⁇ fic for the analyte of mterest
  • This mixture is then allowed to react typically for 1-90 mm du ⁇ ng which the analvte and the labelled analyte equivalent compete for binding to the immobihsed binding spe ⁇ es.
  • the amount of bound labelled analyte equivalent is inversely proportional to the level of the analvte in the sample.
  • the bound electrochemically activatable and/or detectable label or moiety is detected as outlined in other of the two methods given above.
  • An example of such an assav for oestradiol is outlined below in the examples. Scanning or alteration of the pola ⁇ ty and position of the bipolar faces to spe ⁇ fically generate ECL by rapidly generating oxidised and reduced spe ⁇ es (i.e. Ru 3+ and Ru + ) is also contemplated. These are then able to react to yield the ex ⁇ ted state which then emits electromagnetic radiation.
  • Varying the frequency of the apphed voltage from 0 to 200,000 cycles per second, and advantageously from 0 to 5,000 cycles per second to enhance certain reaction mechanisms, is contemplated. This is demonstrated in the double potential step mode of ECL (Edge et al), and in luminol hydrogen peroxide electrochemically activated che ⁇ ulu ⁇ unescence.
  • va ⁇ ous formats give nse to the followmg compositions in the presence of analyte du ⁇ ng the spe ⁇ fic bmdmg step(s);
  • Ab is selected from at least one of the followmg; antibody, nucloc a ⁇ d, mole ilar spe ⁇ es capable of bmding in a sequence-dependent fashion to a nucloc a ⁇ d sequence, a so-called nucloc a ⁇ d analogue or other nucl ⁇ c analogue, or a receptor or hgand.
  • SA is selected from at least one of the followmg; streptavidin, avidin or a molecular spe ⁇ es able to bind biotin or
  • At is selected from at least one of the followmg; the analyte of mterest such as a hgand or nucloc acid.
  • EL is the electrochemically active label.
  • Structure is selected from examples of structures of the subject mvention descnbed earher.
  • Spatial arrays of multiple zones or elements are also contemplated by this invention.
  • This consists of conducting and/or semi-conducting structures of the subject mvention spatially arranged such as on the surface of a non conducting surface such as glass or plastics
  • the methods for putting down or coating multiple elements or zones of conducting and/or semi-conducting materials are well known in the art, e.g. sputtering, evaporation, electron beam evaporation, screen printing, painting, ink jet printing, other printing methods, photocopying, stamping, physically placing elements and fixing them in place, etc.
  • the mterest m generating such spatial arrays of structures of conducting and/or semi-conducting material of the subject invention is to allow for the construction of an analyte-detection device for multiple analytes where each of the structures of conducting and/or semi-conducting matenal is a potential electrode capable of carrying out an assay for a predetermined analyte.
  • the spatial arrays are able to carry out multiple analyte assays without the need for mdividual reaction chambers or tubes, or the need for multiple electromc conducting contacts to the structures of conducting and/or semi-conducnng material.
  • vanous materials can be mixed (see above) te. Pt, Au, Ag, Cu, C (and its vanous forms).
  • vanous methods for making the contemplated structures can be used in combination to achieve the optimal combmations of structures.
  • the use of combinations of coating methods can also be used to develop combinauon mate ⁇ als such as platinized carbon by vacuum- deposition methods for Pt onto screen-printed Carbon structures.
  • vanous sizes of structures of conducting and/or semi-conducting matenal are used to allow the sequential activation of va ⁇ ous elements to provide an addition means for determining which assay is bemg read in addition to the spatial information from the structures' position withm the spatial array
  • This use of va ⁇ ous sizes of structures of conducting and/or semi-conducting material - which will develop the potential required to activate the detectable electrochemistry of the subject mvention - is achieved through the dependence of the structure size and the apphed voltage m determining the voltage developed across a given structure within the apphed voltage gradient
  • a multi arrav device might be constructed with st ⁇ ps of carbon paste, (glassy carbon particles for example from l-2000 ⁇ m in size, m a plastic or resin b der) or conducting composite.
  • the methods used bv Iwasaki may be used to generate dun patterned films of graphitic and conducting carbon (Y Iwasaki, Current Separations (1995) 14, 2-8 and O Nrwa, Electroanalysis (1995) 7, 606- 613) These st ⁇ ps of composite carbon could be va ⁇ ous lengths such as 0.1, 0.2, 04, 0.8, 1 6 mm.
  • the detection and/or quantitation of multiple analytes on a single surface or with a single container is an advantageous embodiment of the subjett mvention.
  • multiple zones or elements are fab ⁇ cated onto a surface followmg conventional methods descnbed for externally electromcally connected lnterdigitated array microelectrodes (Y Iwasaki, Current Separations (1995) 14, 2-8 and O Niwa, Electroanalysis (1995) 7, 606-613) or constructed within a three dimensional structure such as a filter or fabnc.
  • a sample would be introduced mto a container, and within this container multiple zones or elements containing structures of conducting and/or semi-conducting matenal of the subject mvention not electromcally connected to an external electromc circuit would allow multiple assays to be earned out.
  • These assays would be based on the electrochemistry produced by the structures of conducting and/or semi-conducting material of the subject mvention not electromcally connected to an external electromc circuit when subjected to a voltage gradient in a suitable electrolyte. Further applications of this embodiment are desc ⁇ bed below.
  • the multiple zones or elements for these multiple assays consist of other a single structure of conducting and/or semi-conducnng matenal forming the zone, or multiple structures of conducting and/or semiconducting material grouped to form the zone or element in a defined region or portion of an assay component (surface or three dimensional array)
  • an example can be given where gold, carbon or other conducting or semi-conducting matenal is evaporated, sputtered, deposited, printed, painted or formed onto a surface making a defined shape based on a mask or printing device used in the process or by the use of resist patterning and selective etching
  • These defined shapes are electromcally conducting and/or semi-conducting throughout the bed shape and form the zone or element in which an assay for an analyte can be conducted.
  • the multiple structures of conducting and/or semi-conducting material are used to form a zone or element for performing a given assay Examples of this are the same as for the previous example but here, the mask or printing device, or the use of resist patterning and the selective etching used in the process, allows multiple structures to be deposited or formed in a given zone or element Alternatively, the multiple structures of conducting and/or semi-conducting matenal are already made and are simply apphed to the surface such as with carbon fibres, fibrils, nanotubes or particles.
  • vanous methods result in the formanon of multiple zones or elements (each able to carry out an assay) and each zone or element contams multiple structures of conducting and/or semi-conducting matenal
  • va ⁇ ous printing methods known in the art with or without binding material to bond the structures to the surface by physical immobilisation within a mat ⁇ x (such as descnbed m example 15 for the filter wicking assays).
  • multi arrays can also be formed in a three dimensional array, e.g. usmg arrays of fibres incorporating structures of conducting and/or semi-conducnng materials of the subject invention.
  • the structures of conducting and/or semi-conducting material could be woven mto a fabnc to form a filter or wicking material.
  • the incorporation of structures mto a fabnc or filter is another example of physical immobilisation.
  • These fibres can be made of fibre optical matenal allowing the rumination and/or collection of electromagnetic radiation from these conducting mate ⁇ als withm a sample or withm a mat ⁇ x of such fibres.
  • the structures of conducting and/or semi-conducang material can be advantageously coated onto the optical fibres.
  • a surface can be patterned with gold and/or other conducting and/or semi-conducting materials as desc ⁇ bed above usmg va ⁇ ous methods which are known, to form an array of single structures each of which acts as a potential bipolar electrode element for a given assay.
  • the gold forms a single structure of the subject mvention which is also a single zone or region able to be become electrochemically activated by the apphcation of a voltage field m the presence of a suitable electrolyte.
  • the surface can have zones which, rather than havmg a single structure of conducting matenal forming the zones or elements, can be composed of multiple structures withm a defined zone or element for the means of conducting a spe ⁇ fic assay
  • a zone or element for carrying out a given assay might be defined as havmg a size of 5 mm by 5 mm; on a given surface these zones or elements are spaced 2 mm apart
  • the surface would be at least 12 mm by 19 mm with the zones or elements m a 2 by 3 array
  • the 5 mm bv 5 mm zones could be made of evaporated gold, etched carbon, glassy carbon particles or printed carbon nanotubes
  • the 5 mm bv 5 mm zones may be a continuous electromcally conducting structure but could also be composed of multiple structures of 5 ⁇ m bv 5 ⁇ m contamed within the 5 mm by 5 mm zones with spacing of 5 ⁇ m This would create an arrav within the 5 mm by 5 mm zones of structures of conducting matenal
  • a 5 mm by 5 mm zone could contain 250,000 5 ⁇ m bv 5 ⁇ m structures of conducting maten
  • the following example is to further illustrate the detection and/or quantitation of multiple analytes
  • glassv carbon particles there is no need to define the structures of the conducting matenal on the surface as the particles already define the structures of the conducting material.
  • Glassy carbon particles 1% (w/v) m polystyrene (10% w/v) dissolved in chloroform are apphed to a polystyrene surface (15 mm by 20 mm by 2 mm thick) to generate the six 5 mm by 5 mm zones as descnbed above.
  • This surface containing the 5 mm by 5 mm zones, is then etched usmg va ⁇ ous methods to better expose the carbon and activate it for coupling to antibodies usmg va ⁇ ous methods known m the art such as abrasion followed by chemical activation (see examples) or a plasma such as oxygen (O Niwa, Electroanalysis (1995) 7, 606-613).
  • Antibodies are coupled to the oxidised carbon surface as descnbed in the followmg examples. Spe ⁇ fic antibodies are coupled to the 6 zones by spotting the spe ⁇ fic antibody onto each zone. Following coupling of the antibodies to the zones, the polystyrene surface is blocked with a solution of bovine serum albumin and washed ready for use m an assay.
  • the surface is then covered with the sample of mterest and incubated for 1 hr followed by washing.
  • the surface is then incubated with antibodies labelled with Ru(bpy) 3 2+ for 1 hr followed by washing in PBS.
  • the surface is then placed in a cell containing an electrolyte able to support ECL as desc ⁇ bed in the examples, and is subjected to a voltage field.
  • the light emitted from each of the zones is detected usmg a CCD camera.
  • the apphed voltage field is advantageously apphed in a dun film cell such that the surface of the polystyrene coated with the glassy carbon particles is parallel to the voltage field.
  • nucloc a ⁇ d assays In an alternative embodiment, an assay for nucloc a ⁇ ds is contemplated.
  • a specific bmdmg spe ⁇ es for the nucloc a ⁇ d is used, typically a nucleic a ⁇ d sequence but this also could be a nucloc a ⁇ d analogue.
  • nucleic a ⁇ d or DNA of mterest the sample containing the analyte of interest
  • the nucleic a ⁇ d or DNA of mterest can be coupled to the surface of the structures of conducting and/or semi-conducung material usmg methods known in the art via phosphate, amme, aldehyde or thiol groups on the nucloc a ⁇ d.
  • the DNA modified structures would then form the basis of the specific bmdmg surface for the complementary bmdmg nucl ⁇ c a ⁇ d or nucloc a ⁇ d analogue labelled with the electrochemically detectable spe ⁇ es, for example Ru(bpy) 3 2+
  • the DNA modified structures are then subjected to a so-called pre-hyb ⁇ disation reaction which effectively blocks the non-spe ⁇ fic binding sites on the structure of conducting and/or semt- conducting material.
  • the DNA modified structures are then subjected to the spe ⁇ fic hyb ⁇ disation reaction with complementary bmdmg nucloc a ⁇ d or nucl ⁇ c a ⁇ d analogue labelled with the electrochemically detectable spe ⁇ es, for example Ru(bpy) 3 2+ .
  • This hyb ⁇ disation reaction between the structure-bound DNA and the complementary bmding nucl ⁇ c a ⁇ d or nucleic a ⁇ d analogue labelled with the electrochemically detectable spe ⁇ es can last for 1 mm to 24 hrs and depends on the concentration of the complementary bmding nucleic a ⁇ d or nucloc acid analogue labelled with the electrochemically detectable spe ⁇ es, for example Ru(bpy) 3 2+ , the temperature and the ionic strength of the hybndisa ⁇ on buffer known in the art After the hybndisa ⁇ on step, the structures are washed into an electrolyte able to support the detectable electrochemistry of the electrochemically detectable spe ⁇ es.
  • the structures are then introduced mto the cell of an mstrument of the subject mvention and subjected to the voltage field apphed to the central zone of the cell by the externally electromcally connected electrodes.
  • the detectable electrochemical reaction is then detected, for example by a PMT, CCD camera or photodiode in the case of a detectable electrochemical reaction based on ECL or chemilummescence.
  • the detection and/or quantitation of the detectable electrochemical reaction is used to determine the presence and/or quantity of a nucloc a ⁇ d sequence of mterest in the analyte or sample nucl ⁇ c a ⁇ d.
  • the ECL hght level is used to determine the presence of a given DNA sample and/or the number of genes present in the sample.
  • the complementary bmdmg nucl ⁇ c a ⁇ d or nucl ⁇ c a ⁇ d analogue labelled with the electrochemically detectable spe ⁇ es for example Ru(bpy)3 2+
  • a specific nucloc a ⁇ d sequence (or nucl ⁇ c a ⁇ d analogue) for the analyte nucloc a ⁇ d of mterest is coupled to the surface of the structures of conducting and/or
  • the sample nucloc acid containmg the analyte nucl ⁇ c a ⁇ d is added to these pre-hyb ⁇ dised structures other with, before or after the addition of a nucloc a ⁇ d or nucloc a ⁇ d analogue labelled with the electrochemically detectable spe ⁇ es, for example Ru(bpy) 3 2+ , which is similarly complementary to the analyte of mterest, and subjected to hybndisauon
  • the nucl ⁇ c a ⁇ d coupled to the structure and the nucloc a ⁇ d labelled with the electrochemically detectable spe ⁇ es are selected such that they hvb ⁇ dise to the analyte nucloc a ⁇ d in such a way that the analyte nucloc a ⁇ d in the sample links the structure-bound nucl ⁇ c a ⁇ d to the nucloc a ⁇ d labelled with the electrochemically detectable spe ⁇ es
  • the structures are washed mto an electrolyte able to support the detectable electrochemistry of the electrochemically detectable spe ⁇ es
  • the structures are then subjected to a voltage field apphed to the central zone of the cell by the externally electromcally connected electrodes.
  • the detectable electrochemical reaction in the case of a detectable electrochemical reaction based on ECL or chemiluminescence, is then detected for example by a PMT, CCD camera or photodiode.
  • the detecnon and/or quantitation of the detectable electrochemical reaction is used to determine the presence and/or quan ⁇ ty of a nucloc a ⁇ d sequence of mterest in the analyte or sample nucl ⁇ c a ⁇ d.
  • the ECL light level is used to determine the presence of a given DNA sample and/or the number of genes present in the sample.
  • the structures of conducting and/or semi-conducting material are coated, coupled or de ⁇ vatized with a bmdmg spe ⁇ es which bmds to a bmding spe ⁇ es on a nucloc a ⁇ d or nucloc a ⁇ d analogue.
  • streptavidm could be coupled to the structure and the nucloc a ⁇ d coupled to biotin.
  • This method also allows for the use of a universal structure and a spe ⁇ fic capture o gonucleo ⁇ de which provides for greater flexibility in the development of a random access analyser to assay a number of different analytes.
  • This p ⁇ n ⁇ ple forms the basis of the Roche Boeh ⁇ nger Mannheim Elecsys system where the structures (paramagnetic beads) are coated with streptavidin.
  • nucloc a ⁇ d detection which differ from the traditional bmding reaction-based tests are those where the sample containing the analyte nucloc a ⁇ d is subjected to an enzymatic step which allows amplification of the amount of the analyte nucl ⁇ c a ⁇ d m the sample and/or labelling of the analyte nucl ⁇ c a ⁇ d with other a bmding spe ⁇ es or an electrochemically detectable spe ⁇ es
  • PCR polvmerase chain reaction
  • one or more ohgonucleo ⁇ de sequences (p ⁇ mers) are used to prime the synthesis of nucl ⁇ c a ⁇ d resulting m the amplification of the analvte nucl ⁇ c a ⁇ d which is adjacent to the pnmers used in the PCR
  • the product of such a PCR is nucloc a ⁇ d which can be detected and/or quann
  • the p ⁇ mers used to amplify the analyte nucloc a ⁇ d could be labelled with other a bmding spe ⁇ es or an electrochemicallv detectable spe ⁇ es
  • the products of this reaction are detected and/or quanntated by hyb ⁇ disation usmg structures coupled to a nucl ⁇ c a ⁇ d sequence spe ⁇ fic (and complementary) to the analyte nucloc a ⁇ d sequence which is amplified by the pnmer labelled with the electrochemically detectable spe ⁇ es.
  • nucl ⁇ c a ⁇ d-coupled structures are able to be substituted with a combmation of a structure coupled to a bmd g spe ⁇ es (stteptavidin) and a nucl ⁇ c a ⁇ d sequence spe ⁇ fic (and complimentary) to the analyte nucl ⁇ c a ⁇ d sequence labelled with a complementary bmdmg spe ⁇ es (biotin) to the bmdmg spe ⁇ es coupled to the structure, as descnbed above
  • the format j ust desc ⁇ bed could be modified such that the bmding spe ⁇ es is incorporated mto the PCR and the electrochemically detectable spe ⁇ es can be attached to the nud ⁇ c a ⁇ d sequence specific (and complementary) to the analyte nudoc
  • nudoc a ⁇ d usmg p ⁇ mers are also used to label nudoc a ⁇ d analytes of mterest with bmding spe ⁇ es and/or electrochemically detectable spe ⁇ es usmg the pnmer.
  • labelling of nud ⁇ c a ⁇ d with va ⁇ ous spe ⁇ es is possible via the use of direct incorporation of labelled NTPs and chemical modification of the nud ⁇ c a ⁇ d (US5512433).
  • the sample nudoc a ⁇ d is hyb ⁇ dised to a nudoc a ⁇ d or nud ⁇ c a ⁇ d analogue which is able to form an antigen sigmficandy different from other nucl ⁇ c a ⁇ d in the hyb ⁇ dised form such that an antibody will bmd the hyb ⁇ d and allow assay of the analyte nudoc a ⁇ d.
  • the analyte can be labelled with va ⁇ ous spe ⁇ es and/or the nud ⁇ c a ⁇ d and/or the antibody specific for the analyte nudoc a ⁇ d to probe the nudoc a ⁇ d hyb ⁇ d which is formed.
  • nud ⁇ c a ⁇ d sequence which is able to hyb ⁇ dise to an analyte nud ⁇ c a ⁇ d other direcdy from a sample or after amplification using for example PCR, NASBA or other target-amplification systems.
  • a probe sequence labelled with an electrochemically detectable spe ⁇ es complementary to the structure-coupled nud ⁇ c a ⁇ d is contemplated.
  • the analyte nud ⁇ c a ⁇ d competes with the bmdmg of the probe sequence labelled with an dectrochemically detectable spe ⁇ es for the structure-bound nud ⁇ c a ⁇ d.
  • a loss of structure-boimd electrochemically detectable spe ⁇ es results from hybndisation to the analvte nud ⁇ c a ⁇ d This results m a signal decrease.
  • An obvious modification to this method would be to use a bmding spe ⁇ es on the structure (streptavidm) and a bmding spe ⁇ es on the capture nudoc a ⁇ d (biotin) such that the nucl ⁇ c a ⁇ d is captured onto the structure at the desired time.
  • the hyb ⁇ disation steps, wash steps and analysis are as desc ⁇ bed m the examples above
  • sample containing analvte nudoc a ⁇ d of mterest indude cells, biological fluids such as serum, plasma, urine, saliva, stool, semen, sputum, cell lysates, viruses, bactena, plant tissue, animal nssues, fungi, achea bacte ⁇ a, DNA, cDNA, RNA, mRNA, tRNA, rRNA, plasmids, phage, mitochondria! nud ⁇ c a ⁇ d.
  • a flow-through assay system is contemplated. This method is related to methods used for cell counting and fluorescent cell analysis but provides a novd solution to improving assay sensitivity and simph ⁇ ty.
  • an mstrument is constructed which contams the basic dements descnbed above containmg a voltage control ⁇ rcuit designed to apply a voltage usmg the externally connectable dectrodes which are contamed in a cell, a detector for dectromagnenc radiation which is a photomul ⁇ pher tube (PMT), a computer for the control of the voltage circuit, a detector and a pump.
  • PMT photomul ⁇ pher tube
  • the mstrument contains a pump which controls - with the aid of the computer - the flow of the sample, and a cell which is designed to have a narrow zone (the central zone of the subject mvention) through which will flow the structures of the subject mvention which are advantageously conducting partides.
  • the cell of this mstrument m its basic form could be a simple tube with a neck or narrow portion such that the flow of sample containing the conducting particles results m the separation of the particles allowing mdividual partides to be present m this neck or narrow zone.
  • This basic cell would have externally connected electrodes positioned other side of the central zone composed of the neck or narrow portion of the flow cell.
  • an assay would be constructed with partides of vanous sizes such that each bmding spe ⁇ es for each assay would be bound to a different size parade.
  • Each group of different size particles would be activated at different positions in the flow cell as the sample flowed through under the voltage apphed from the externally connected dectrodes.
  • This mixture of vanous size particles coated with a number of different bmding spe ⁇ es would be mixed with a sample of mterest and with labelled bmdmg spe ⁇ es to effect a bmdmg of the labelled bmdmg spe ⁇ es proportional to the concentration of the analytes of mterest m the sample of mterest.
  • each size of conducting parade would be coated with a different antibody. This would result in a mixture of parades of va ⁇ ous sizes where a given parade size would have a given antibody coated on it
  • This mixture of coated conducting particles would then be added to a sample which contams a senes of analytes to which the antibodies coated on the conducting particles could bmd
  • labelled antibodies or labelled anngen labelled with an electrochemilummescent or chemiluminescent spe ⁇ es
  • This mixture of coated conducting particles, sample and labelled antibodies and/or antigens would then be allowed to bmd.
  • the sample will generate light from the vanous size parades at spe ⁇ fic positions in the flow cell path depending on the size of the parade
  • the location of the light is determined by the use of light detectors positioned such that light is detected at ed positions m the flow path This detection of light can be achieved advantageously by the use of a diode arrav or similar as descnbed earlier.
  • the light emitted is opttonally subjected to spectral analysis.
  • This flow-through assay system based on conducting partides thus offers great value as it provides multiple analyte detection and/or quantitation using parade size.
  • This advantage of the flow-through system is enhanced by its ability to carry out multi-analyte detection usmg multiple wavdengths
  • each parade's light emission is analysed spectrally, and a spectral signature is all that is required to identify and thus assay a given parade Smce each parade is spectrally analysed as it passes the detectors, labds with overlapping spectra are readily detectable using, for example, in its simplest form, a 2 spectral window detection system.
  • Such a system based on two spectral windows can readily detect multiple labds with minor differences in peak emissions when the labds are ex ⁇ ted and analysed separately
  • Such a system has been used to detect the presence of at least 4 labels in a DNA sequencing system produced bv DuPont In the case of the system used by Luminex 64, different beads are detected based on two fluorescent dyes, one orange and one red.
  • An alternative cell configuration consists of electrodes which flank the narrow portion of the flow cell with a voltage apphed across the path of the particle as it flows through the cell
  • detection of the parade is optionally incorporated mto the mstrument
  • a number of methods could be used based on methods currendy employed for particle analysis or cell analysis
  • An example of this is the svstem used m the copahs technology (Sienna Biotech, Columbia MD) where size is determined by light scattering Examples of such methods which could be advantageously incorporated m the flow-through system are given hereafter.
  • Light or advantageously a laser could be used to detect and analyse the parades as thev pass through the detection zone.
  • a light source to the flow-through mstrument may not require a supplementary dete ⁇ or but could make use of the existing detector used for the ECL emission.
  • this hght source could be pulsed at a given frequency such that the ECL and fluorescent light emission could be multiplexed and then (discriminated by the light detector and analysis system.
  • the advantage of this system is that it would provide information concerning the size and spectral properties of the structure of conducting and/or semi-conducting matenal of the subject mvention and correlate this with the ECL for the parade. Thus, this system would allow for multiple analyte detection and/or quantitation. Based on results obtained with the Luminex system, 64 to 500 assays may be readily earned out with this type of system.
  • An alternative embodiment of the subject invenuon wherein structures of the subjett invenuon coupled to binding spe ⁇ es are used to bmd to cells resulting m aggregation or the formation of multiple beads around a cell or multi-cell collection such as an embryo or micro-organism is contemplated. These aggregates of more than one bead would then be detected based on the intensity of the light emitted as they pass through the devated voltage zone which acnvates the dectrochemisrry on the surface of the structures.
  • this analysis would make use of the structures as labds and most advantageously the detectable dectrochemisrry would be ECL.
  • the cells would be added to a buffer containing an ECL speaes and a coreactant if needed to allow ECL at the surface of the structures as they pass the activation and detec ⁇ on zone.
  • the structures used in this embodiment would be beads, parades, nanotubes or fib ⁇ ls. More advantageously, the structures would be nanotubes or fibnls. Most advantageously, the structures would be graphitic carbon nanombes or fib ⁇ ls
  • analytes are detected form duates of gels, HPLC, FPLC, chromatography and dectrophore ⁇ c separation methods.
  • a flowing stream from the separation method would be mixed with a flowing stream of buffer, containing structures of the subject mvention.
  • the results of the activation of electrochemistry would generate a detectable signal
  • An example could be the detection of amines from HPLC as has been descnbed (Forbes et al 1997, Analvtica Chimica Acta 347, 289- 293) usmg conventional dectrochemical methods.
  • a flowing stream of Ru(bpy) 3 2+ with the structures of the subject mvention would be mixed with the HPLC duate and then passed through a cell where the voltage gradient is apphed as described above.
  • the structures would be carbon fibres or nanombes.
  • the structure is a carbon nanotube or fibril (advantageously 0.02 ⁇ m to 2 mm, most advantageously 1 to lOOO ⁇ m in length) which is advantageously labelled with a binding spedes such as an antibody, nudoc add, or receptor hgand.
  • a binding spedes such as an antibody, nudoc add, or receptor hgand.
  • This binding spedes-attached carbon nanotube or fibril is then used as a reagent in binding to samples of binding spedes present on a surface.
  • This may be the surface of a synthetic material but advantageously the surface is a biological material grown or deposited onto a surface such as a glass microscope slide.
  • tissue sections, cell cultures, immobilised arrays of nudoc acid, smears of tissue which are processed to preserve the biological material of interest, stabilise the biological material and improve the presentation of the biological material
  • the surface is washed thoroughly and placed in the central zone of a cell which contains ECL reagents that may be in organic solvents.
  • the ECL reagents are other tnpropylamine and Ru(bpy) 3 2+ or hydrogen peroxide and luminol or luminol, hydrogen peroxide and ferrocene.
  • This cell which contains the two externally connectable dectrodes flanking the central zone, is place in an instrument as described earlier for the apphcation of the external voltage and detection of the ECL light emission.
  • the use of a microscope and CCD camera has certain advantages in the instrument in that this provides a spatial determination of the position of the bound carbon nanotubes or fibrils on the surface of the sample.
  • This use of the subject invention allows for sensitive detection of binding spedes on a surface and also provides the potential to determine spatial information concerning the distribution of a given bmding spedes on a cell or within a celL or the counting of the given cell type within a population. Assays based on structure to structure binding
  • assays based on the binding of at least two structures of conducting and/or semi-conducting material is also contemplated.
  • Such assays consist of detecting the formation of induced proximity of two conducting and/or semi-conducting material by methods of the subject invention.
  • a binding spedes and/or pair of binding spedes are coated onto structures of conducting and/or semi-conducting material such that they are able to bind a given analyte in such a way that at least two structures of conducting and/or semi-conducting material are linked via the binding to the analyte.
  • This formanon of a redox couple allows for effective channelling of reactants from the anode face of one parade to the cathode face of the other, thus allowing the generation of an dectrochemical reaction only effectivdy seen when the at least two parades are linked in this way
  • An example of such a coupled reacnon would be where ECL is generated from the oxidised and reduced spe ⁇ es of ECL labels, te. when Ru + and Ru 3+ react they generate light.
  • an assay would allow bmdmg to be caused by the bmding spe ⁇ es and the bmding of the structures of conducting and/or semi-conducnng matenal in proximity would allow the detection of the bmding reaction via the induction of EC of the subject mvention.
  • induction of EC would occur m the presence of an ECL spe ⁇ es as illustrated above
  • numerous methods can be used to achieve the linking or mduce proximity of structures of conducting and/ or semi-conducting material.
  • the structures may be coated or linked to an enzyme substrate which when acted on by an enzyme results m the linking or mduced proximity of the coated structures.
  • an assay for the enzyme and/or its substrate may be constructed.
  • Enzyme assays using structure-bound labels The assays descnbed are directed at the detection of bmding reactions, or the detection of a soluble analyte direcdy or generated by a chemical or enzymatic reaction. It will also be understood that other chemical and enzymatic assays can also be earned out using a number of the formats outhned above These chemical and enzymatic assays would be based on both synthesis reactions and degradation reactions For example, a synthesis reacnon would be the action of DNA polymerase on a template nud ⁇ c a ⁇ d initiated by a pnmer.
  • the pnmer could be labelled with an dectrochemicallv detectable spe ⁇ es such as an ECL labd and du ⁇ ng the polymerase reaction a bmding spe ⁇ es could be mcorporated such as biotin usmg a biotinylated NTP
  • a polymerase reaction would be a nucloc a ⁇ d strand containing both a bmding spe ⁇ es and an dectrochemically detectable spe ⁇ es.
  • This nud ⁇ c a ⁇ d strand could then be captured onto a structure of the subject invention and analysed to determine the levd of polymerase activity
  • synthetic enzymes are DNA, RNA polymerase, glyco-transferases, terminal transferases.
  • the substrate could be coupled to the structure of the subject mvention m such a way that it contams an dectrochemically detectable spe ⁇ es which is removable by the action of the spe ⁇ fic protease or hydrolase
  • modified structures are then incubated with the protease or spe ⁇ fic hydrolase.
  • Incubanon m the presence of an enzyme able to hydrolyse the substrate, and thus remove the electrochemically detectable spe ⁇ es, results m a loss of signal.
  • hydrolases are glycosidases, nudeases, protease and pases which may be used in assays as above. It will be understood that other the enzyme or the substrate may be assayed using the methods outlined above.
  • a further embodiment of the subject mvention is a new way of generating hght from an dectrochemical reaction usmg for example compounds from Maness KM et al J. Am. Chem. Soc 1997, 119, p3987 (see above reference m ECL labds).
  • the structures of conducting and/or semi-conducting materials of the subject mvention are mixed with for example [Ru(bpy) 2 (bpy(C ⁇ 2MePEG350) 2 ](Cl ⁇ 4)2 in for example a methanol water mixture and drop cast onto a non conductive surface such as glass or Si/Si ⁇ 2 between two dectrodes formed on the glass or Si/Si ⁇ 2 using conventional methods.
  • Example 1 Example of ECL apparatus and a method for obtaining a detectable signal from dectrolyte-suspended conducting partides.
  • the apparatus integrates a computer-controlled programmable voltage source (Kdthley 230), a photomultiplier (Hamamatsu H5323) connected to a photon counter, a digital multimeter (Philips PM 2422A) and a custom-made cell.
  • the cell is the heart of the instrument and is illustrated in frontal view in schematic 1, in top view in schematic 2 and in side view m schematic 3.
  • the cell is constructed from a block of PVC 1 mto which is cut channd 2. Channd 2 contams two platinum dectrodes 13 and 14 fixed to other end.
  • Two holes 3 and 4 are drilled mto each side of PVC block 1 for platmum electrodes 13 and 14 to be externally dectromcally connected via connectors 5 and 6 to the voltage source through leads 7 and 8.
  • Two holes 9 and 10 are drilled perpendicular to channel 2 for insertion of a fibre optic tube 16.
  • a lens 15 is placed between the end of fibre opnc tube 16 and channd 2 in such a manner than fibre optic tube 16 views soldy the central poraon of channd 2.
  • a PVC hd of the same dimensions as PVC block 1 fits onto two lugs 11 and 12 situated on the upper surface of PVC block 1 to prevent extenor light from entering channd 2.
  • the test sample is placed in channd 2 and a voltage is apphed via dectrodes 13 and 14 across channd 2.
  • light generated withm the sample is detected by fibre optic tube 16 and earned to the photomultiplier.
  • the data from this photometer is digitised and analysed by a computer (Dell Corporation XPS 200, software: National Instruments, Aus ⁇ n Tx).
  • the voltage apphed to the two external plaunum dectrodes (from a Kdthley 230 programmable voltage source) is controlled by the computer in such a way that the voltage apphed to the channd du ⁇ ng a measurement cycle is corrdated with the light level detected at the PMT.
  • the voltage control is constructed to prevent arcing with high current.
  • the general layout of the experimental mstrument is illustrated m schematic 4 where 17 is the voltage generator, 18 is the digital multimeter set to monitor the current, 19 is one of the leads connecting the voltage source to the platinium dectrodes, 20 is the reaction cell, 21 is the fibre optic tube, 22 is the luminometer/PMT and 23 is the controlling computer
  • This mstrument is designed for multiple cell configurations to be used. Cell l
  • One cell has a continuous channel from one end to the other which allows samples to be mtroduced using a pump or other flow control method.
  • the two externally dectromcallv connected dectrodes are attached to the channd via a porous fnt, permeable or semi-permeable diaphragms or membranes such that electncal contact is made through an electrolyte to the channel which forms the measuring zone.
  • This cell configuration allows samples to be placed in the measuring zone and then ex ⁇ ted via the apphed dectnc fidd between the two externally electromcally connected dectrodes.
  • the second cell (illustrated m schematics 1, 2 and 3) is configured as in the first cell with respect to the externally dectronically connected dectrodes but does not have a continuous channd from one side to the other. Instead, the sample is mtroduced via the top of the channd cut mto the PVC block.
  • the externally dectromcallv connected dectrodes are isolated from this channd via a porous fnt or semi-permeable membrane This cell allows manual washing and introduction of the sample followed by a measurement cyde Cell 3
  • the third cell is designed as for the second cell but the central channd is modified such that it is able to accept or hold a filter paper or immunochromatographic filter element or gel.
  • This channel is able to accept the filter paper or immunochromatographic filter element or gd in such a wav that dectrolyte is in contact with the filter paper or immunochromatographic filter dement or gd and with the externally electromcally connected electrodes
  • This channd with the filter element also has a window to hold the filter element in place and to allow dose proximity of the PMT or fibre optic tube to the filter element, improvmg photon capture by the PMT.
  • the window dement is vanously glass, plastic, and plastic wrap which is transparent to the photon wavdengths of mterest
  • the above mstrument is also configured to allow the use of a photodiode in place of the PMT
  • a suspension of chopped carbon fibres lOOO ⁇ m x 7 ⁇ m (Goodfellow Cambndge) at 3-15 ⁇ g/ml in ECL buffer (100 mM tnpropylamine, 002% (w/v) Tween 20, pH adjusted to 7 2 with 85% orthophospho ⁇ c acid) and 10-lOOnM Ru (2 ⁇ -b ⁇ pv ⁇ dvl) 3 2+ [Ru (b ⁇ y)3 + ] was introduced manually mto cell 2 through the top of the channel.
  • the electrodes at each end of the channd were then connected to the voltage source (Kothley 230 generator) and the fibre op ⁇ c tube was inserted mto the cell to view the central portion o the channd m which the electrochemistry occured.
  • a ramp voltage from 0 to 100 V was then apphed to the platinum electrodes for 20 seconds.
  • the PMT (Hamamatsu H5323) monitored the hght produced du ⁇ ng the ramp and the current flow was monitored using a digital multimeter (Philips PM 2422A).
  • the dectrochemistry, and therefore an analyte can be detected other amperomet ⁇ cally through the determination of the resultant current with respect to the apphed voltage potential, spectrophotometncally by the production of a new chromophore, or through the generation of ECL.
  • Tests usmg different reagent concentrations showed that the ECL signal detected by the PMT mcreased with mcreasmg numbers of fibres and with increasing concentrations of Ru (2 ⁇ -b ⁇ py ⁇ dyl) 3 2+
  • Ru (2 ⁇ -b ⁇ py ⁇ dyl) 3 2+ Example 2
  • Carbon matenal suspensions as desc ⁇ bed in example 2 are added to PBS to generate a 0.1-1 mg/ml suspension of carbon. These are washed once then antibody or streptavidm is added at a concentration of 100-300 ⁇ g/ml in a pH 9 6 100 mM sodium carbonate/bicarbonate buffer. The fib ⁇ l mix is left to mix at 48°C for 16-20 hours. Followingmg this incubation, the fib ⁇ ls are washed by centrifugation and resuspended in 3% BSA, 0.1% Tween 20 PBS and incubated for 2 hours to block any uncoated sites.
  • fib ⁇ ls are then washed 5 times m the 3% BSA, PBS with 0.05% sodium azide and finally resuspended in this buffer for storage. Pnor to use, the fib ⁇ ls are washed mto PBS 0.1% Tween 20 to a concentration of 0.1-1 mg/ml. In the case of other carbon parades, these are prepared by a quick wash m PBS followed by addition of the an ⁇ body-containmg solution and apphcation of the protocol above.
  • Carbon parades, fibres and fib ⁇ ls are obtained from MER corporation and Alfa Aesar (Ward HilL MA). These are activated using oxidative methods such as perchlo ⁇ c a ⁇ d, chromic a ⁇ d treatment, oxygen plasma or other methods known in the art to generate -COOH functional groups on the surface of carbon, glassy carbon and graphitic carbon (see example 2). These carbon surfaces - de ⁇ va ⁇ zed with COOH groups - are suspended in anhydrous dioxane with mixing to concentrations of between 0.1 and 10 mg/ml.
  • d ⁇ ed NHS-ester parades are washed with PBS and added to a solution of the biomolecules to be coupled, typically streptavidin, an ⁇ bodv and ammo-modified DNA or ohgonudeotide.
  • streptavidin a 5-10 mg/ml solution in PBS is used.
  • ohgonudeo ⁇ de between 15 and 40 bases long is used.
  • the parades and biomolecules are mixed to form a suspension and this is then incubated for 1-2 hours at room temperature.
  • Example 5 Labelling of protems with ECL labels 1 mg of protem is labelled with Ru (2,2-b ⁇ pyndyl) 3 2+ (Ru(bpy) 3 2+ )
  • the protein is buffer-exchanged usmg Cent ⁇ con 30 microconcentrators (Amicon) mto 0 15M potassium phosphate buffer, 0 15M NaCl pH 7.8, the final volume bemg 0.5 mL
  • 0 5 mg of Ru(bpy)-s 2+ -NHS (Ru(2,2-b ⁇ py ⁇ dyl)2(4-[3- (l,3-d ⁇ oxolan-2-yl)propy ⁇ ]-4-metnyl-2,2-b ⁇ pyn ⁇ ine) 2+ ) is dissolved with 125 ⁇ l of anhydrous dimethyl sulfoxide (Aldnch).
  • a molar ratio of about 25.1 should be achieved between the Ru(bpy) 3 2+ and the protein, based on molecular weights of 1057 for Ru(bpy) 3 2+ -NHS and 150,000 for antibodies.
  • the Ru(bpy) 3 2+ -NHS (45 ⁇ l) is added to the protem solution while shaking.
  • the ranges for labelling can be from 3:1 to 30:1 molar ratios depending on the protem bemg labelled.
  • the reaction tube is mcubated in the dark at room temperature for 30 minutes while shaking. The reaction is terminated by the addition of 25 ⁇ l of 1M gly ⁇ ne and ln ⁇ iba ⁇ on for 10 minutes.
  • the reaction mixture is punfied by passage through a Sephadex G-25 column (1 X 20 cm in 0.15M potassium phosphate, 0.15M NaCl with 0.05% sodium azide pH 7.2).
  • the Ru(b ⁇ y)3 2+ - labelled protem fractions are collected and pooled. These labelled proteins are then diluted mto PBS buffer with azide to give protem concentrations between 1.6 ⁇ g/ml and 32 ⁇ g/ml.
  • Immunoassay for the quantitative determination of human cho ⁇ onic gonadotropin This electrochemilu ⁇ unescent assay demonstrates the p ⁇ n ⁇ ples of the subject mvention. The measurement of HCG concentrations can be used to diagnose pregnancy just one week after conception.
  • HCG STAT immunoassay reagents are obtained from Boeh ⁇ nger Mannheim, ⁇ .e. HCG STAT immunoassay reagents (Cat #1731 289) suffi ⁇ oit for 100 tests.
  • This test kit is used to provide the two antibodies, one being labelled with an dectrochemically detectable spe ⁇ es (Ru(bpy) 3 2+ ), the other bemg a bifunc ⁇ onal bmding spe ⁇ es composed of an antibody conjugated to biotin.
  • the sohd phase corresponds to the glassy carbon beads prepared m examples 3 and 4 and coated with streptavidm.
  • the HCG STAT cahbrator set (Cat # 1731670), the buffer to provide optimal ECL, i.e.
  • ProCell (Cat # 1662988) (this buffer is equivalent to the ECL buffer used in example 1 and to ORIGEN assay buffer) and Elecsys Diluent Umversal (Cat # 1732277) are obtained from Boehnnger Mannheim.
  • the samples are prepared from the HCG STAT cahbrator set bv dilution in Elecsys Diluent Umversal
  • One of the calibrators m the HCG STAT cahbrator kit consists of about 10 mlU/ml and the other about 5000 mlU/ml
  • the highest cahbrator is used to make a se ⁇ es of samples bv senal dilution in the Elecsys Diluent Umversal to give a sequence of HCG values which form the basis of the demonstration.
  • the result of the dilutions is a se ⁇ es of HCG values 5000 (from the o ⁇ ginal cahbrator), 1250, 625, 156, 78 and 20 mlU/ml.
  • the assay is performed as follows, 40 ⁇ l of the diluted cahbrator desc ⁇ bed above is added to 220 ⁇ l of the biotinylated antibody and 220 ⁇ l of the Ru(bpy) 3 2+ -labelled antibody This is mixed and mcubated for 5-10 mm followed by addition of lOO ⁇ g (100 ⁇ l) of streptavidin-coated 10-lOOO ⁇ m glassy carbon parades as prepared in examples 3 and 4 This mixture is mcubated for 5-10 nun with mixing This mixture of assav components is then centnfuged and resuspended in the ProCell buffer and washed again before final resuspension in 600 ⁇ l of the ProCell buffer.
  • Measuring oestradiol concentrauons is important in the determination of fertility disorders de ⁇ ved from the hypothalamus-pituitary-gonad axis, gynecomastia and tumours.
  • Reagents are obtained from Boeh ⁇ nger Mannheim Oestradiol immunoassay reagents (Cat #1776002) which contams reagents for 100 tests.
  • This test kit is used to provide the two bmding spe ⁇ es for the assay: oestradiol with a pep ⁇ de linker labelled with an electrochemically-detectable spe ⁇ es (Ru(bpy) 3 2+ ), an antibody used as a bifunc ⁇ onal binding spe ⁇ es and another antibody conjugated to biotin.
  • the sohd phase corresponds to the glassv carbon beads prepared m examples 3 and 4 and coated with streptavid .
  • the Oestradiol CalSet (Cat # 1776037), the buffer to provide optimal ECL, ProCell (Cat # 1662988) and Elecsys Diluent Umversal (Cat # 1732277) are obtained from Boeh ⁇ nger Mannheim.
  • test samples are prepared from Sigma reference standard oestradiol (Cat E 1132) made up in Elecsys
  • the assay is performed as follows; 100 ⁇ l of the sample are added to 180 ⁇ l of the bio ⁇ nylated polydonal anti-oestradiol antibody from the kit and mcubated for 10-15 mm followed bv addition of 180 ⁇ l of the oestradiol-pep ⁇ de hnker-(Ru(bpy) 3 2+ ) and 100 ⁇ l of streptavidin-coated glassy carbon parades (lOO ⁇ g of 10- lOOO ⁇ coated glassy carbon parades prepared as m examples 3 and 4) This mixture is mcubated for 10-15 mm with mixing This mixture of assay components is then cent ⁇ fuged and resuspended in the ProCell buffer and washed again before final resuspension in 600 ⁇ l of the ProCell buffer These samples in the ProCell buffer are then mtroduced mto the cell of the mstrument as desc ⁇ bed in example 1 and the light given off for each sample is determined.
  • TSH assay lOO ⁇ l serum calibrators for TSH 25 ⁇ l of ECL-labelled mouse anti-TSH prepared as in example 5 (typically the amount of antibody added to a test is between 40 ng and 800 ng) and 25 ⁇ l (between 3 and lOO ⁇ g) of glassy carbon structures (10-1000 ⁇ m) coated with sheep anti-TSH in ECL buffer followmg the protocols given in examples 3 and 4, are combined and mcubated in polypropylene tubes for 15 minutes at room temperature with mixing The glassv carbon structures are then washed twice bv cent ⁇ fugation and resuspended in ECL buffer to a final volume of 1 ml These samples are then analysed for ECL usmg the instrument as desc ⁇ bed in example 1 The ECL signals from the va ⁇ ous samples are then plotted against the serum ca
  • Ohgonudeotides are obtained from Ohgo etc (Wilsonville, OR, USA) Ammo-modified ohgonudeotides are labelled with Ru (2,2-b ⁇ pvndyI) 3 2+ usmg the NHS ester or made direcdy during synthesis usmg Ru (2,2- b ⁇ py ⁇ dyl)3 2+ phosphoramidite obtained from IGEN Inc Beta-actin PCR, nucl ⁇ c a ⁇ d probe assav
  • the PCR detection of beta-aeon mRNA is achieved by the conversion of mR mto cDNA followed by a PCR which mcludes a bio ⁇ nvlated pnmer and an ECL labelled pnmer
  • the ECL labd used is ORIGEN TAG (IGEN, Gaithersburg MD)
  • the followmg p ⁇ mers are used to amplify the beta-actin cDNA bv PCR, 5' Biotin-GCC ACA GGA TTC CAT ACC CAA-3' and 5'ORIGEN TAG-GAG AAG AGC TAT GAG CTG CCT GAC-3'
  • mice hver total RNA From 0 1 to 5 micrograms of mouse hver total RNA, including controls not containmg any beta-actin, are reverse-transc ⁇ bed m a 20 ⁇ l reaction for 1 hr at 42°C with Supersc ⁇ pt II RNase H- reverse transc ⁇ ptase, ohgo dT, and lOmM dNTP mix as recommended by the manufacturer (Life Technologies) to generate the cDNA
  • One microhtre of cDNA is amplified in a 100 ⁇ l reaction containmg 1XPCR buffer (Perkin Elmer), 200 ⁇ M dNTPs (LIT), 20 pmoles of each pnmer, and 4 units of Amphtaq DNA polymerase (PE) pre-mixed 1 1 with TaqStart Antibodv (Clontech) Amplification is performed on a Perkin Elmer Model 480 Thermal Cvder usmg the followmg conditions 10 mmutes at 50 C C, 10 mmutes
  • the mixture is rendered lOO ⁇ M ferrocene, lOOuM luminol and 0.1 M Tns buffer.
  • 2-80ug of glassy carbon parades Alfa
  • the samples are then placed mto the ECL mstrument desc ⁇ bed in example 1 followed by ex ⁇ tanon and detection of the ECL light
  • the samples show an increasing signal with increasing cholesterol, forming the basis of a cholesterol assay.
  • Stock solutions of cholesterol are prepared by dissolving cholesterol m a mixture of Tnton XI 00 and ethanol (3:2 v/v).
  • the parades used m this assay are untreated 10-lOOO ⁇ m glassy carbon beads.
  • Example 11 Glucose assay The basic reagent mix for the glucose assay contams 16.8 ⁇ g/ml hexokinase, 20 ug/ml glucose-6-phosphate dehydrogenase, 1 mM ATP, 1 mM NAD + , 2 mM MgSO* and 25 mM T ⁇ s HCL, pH 7 5. Ahquots of this reagent mix are prepared (270 ⁇ l) and 30 ⁇ l of va ⁇ ous glucose standards are added to give a final concentration range of 5 to 35 ⁇ M. These samples are mcubated for 10 mm at room temperature.
  • This solunon is rendered lO ⁇ M with respect to ruthemum (II) t ⁇ s (bipy ⁇ dyl) (Ru(bpy)3 2+ ).
  • This mixture is used as the buffer for the detection of betalactamases.
  • E. coh beta lactamase RTEM To 1 ml ahquots of this mixture are added E. coh beta lactamase RTEM to a concentration of between 1 and 10 nM.
  • the enzyme reaction is allowed to proceed for about 10 min at room temperature (about 20°C).
  • 2-40 ⁇ g of glassy carbon parades (Alfa) are added and the mixture is placed m the central zone of the cell of the mstrument descnbed in example 1.
  • ECL signal is sigmficandy higher in the presence of the beta-lactamase enzyme. This allows a method to be developed for the detection of beta-lactams and betalactamases.
  • the particles used in this assay are untreated 10-lOOO ⁇ m glassy carbon beads.
  • Carboxylate-modified carbon structures prepared as m example 2 (2.5 ⁇ g) are added to 1.0 milhhtre (ml) of methyl ethyl sulfonate (MES) buffer (5 milhmolar (mM), pH 4.75) and 75 micrometres of antibody solution (antibody to beta-hCG, Biogenesis UK) (2 milligrams per milhhtre (mg/ml)).
  • MES methyl ethyl sulfonate
  • EDAC 1 -Ethyl 3(3-D ⁇ methyl-am ⁇ nopropyl) carbodimide HCl
  • antibody, antigen, hgand, receptor and nud ⁇ c a ⁇ d bmdmg sequences may be attached to the parades by a va ⁇ ety of methods, e.g., adsorption or use of va ⁇ ous chemical activators.
  • the partides can be added to the fibrous mat ⁇ x after, for example, animal sera or other blockmg agents have been added, and that the use of such sera is not of cntical importance. Therefore, the order of addition of the parades to the matnx and treatment thereof after or before incorporation mto the mat ⁇ x is not cntical to the present mvention.
  • fibrous mate ⁇ als can be used in place of the glass filter matnx matenal specifically desc ⁇ bed heron, and the advantages of the mvention can also be realised thereby.
  • the glass fibre material containmg the antibody-coated structures as previously desc ⁇ bed (example 14), is cut mto substantially circular "disks", and the disks - forming reaction matnces - are placed m contact with a blotter matenal to absorb excess fluid from solutions used m the assav. Thereafter, five drops of test samples of human u ⁇ ne (about 280 microhtres) containmg zero, and 50 and 100 mlU/ml levels of beta-hCG are added to each matnx after passage of the sample drops through a prefilter situated above each mat ⁇ x.
  • each mat ⁇ x is mcubated at room temperature for about two mmutes.
  • the prefilter is then removed and 1 0 ml of PBS, 0 1% Tween 20 wash solution is added to each matnx to remove an excess an ⁇ bodv-enzyme con j ugate
  • One drop of ProCell buffer is then added to each matnx. After two mmutes, each mat ⁇ x is placed in a cell (example 1, cell 3) containmg two externally and dectromcallv connected dectrodes (see above descnp ⁇ on of ECL instruments).
  • the mat ⁇ x placed in the cell forms an dectncal contact between the externally connected electrodes via the ProCell buffer which forms the electrolyte.
  • Each matnx is then sub j ected to an externally apphed voltage This voltage is apphed at such a voltage gradient up to 10 kv/cm that at least some of the structures trapped in the glass fibre filter are rendered bipolar
  • These bipolar structures activate the ECL from the captured antibody labelled with an ECL active spe ⁇ es and hght is dete ⁇ ed for the test samples which contams beta-hCG The amount of light is correlated to the level of beta-HCG in the sample.
  • the membranes are then washed with a solution of 0.1 mM lummol, 1 mM hydrogen peroxide, 0.1 mM ferrocene monocarboxyhc a ⁇ d (Sigma) and 0.05M T ⁇ s buffer pH 8.
  • the membranes are then trimmed to remove the lower and upper portions of the stnp to leave the site of stteptavidin st ⁇ p.
  • the streptavidin st ⁇ p poraon is placed mto a cell designed to provide dectrolyte contact to two externally electromcally connected dectrodes attached to a voltage control ⁇ rcuit contamed withm the mstrument as descnbed m example 1.

Abstract

L'invention porte sur un procédé de quantification ou détection d'un analyte au moyen d'une réaction électrochimique consistant à mettre dans une cellule ou une chambre un matériau en contact avec un électrolyte, mais sans contact avec un circuit électronique extérieur, une partie au moins dudit matériau étant conductrice ou semi-conductrice, puis à appliquer un gradient de tension sur une partie au moins dudit matériau. La taille du matériau, la conductivité dudit électrolyte et/ou du réactif liquide, et le gradient de tension sont tels que non seulement se créent des faces anodiques et cathodiques sur au moins une partie du matériau conducteur ou semi-conducteur, rendant ainsi ledit matériau bipolaire, mais encore que les électropotentiels ainsi créés sont tels que ladite réaction électrochimique se produit sur au moins une partie de la surface dudit matériau, d'où la production d'un signal détectable ou d'un matériau détectable. Ledit matériau bipolaire qui se compose de préférence de sphères ou de fibres peut consister en un matériau plein conducteur ou semi-conducteur, ou en noyaux d'un matériau non conducteur revêtus d'un matériau conducteur ou semi-conducteur. Le matériau, de préférence en suspension, peut également être fixé ou capturé directement ou indirectement sur une surface ou une matrice filtre. Ladite surface ou matrice si elle est conductrice n'agit pas comme électrode d'électrochimie. La détection du signal ou du matériau obtenu par électrochimie est fonction de la quantité ou de la présence dudit analyte.
PCT/EP1999/003756 1998-06-03 1999-05-31 Procedes, instruments et marqueurs relatifs a des essais electrochimiques WO1999063347A2 (fr)

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EP98430015A EP0962773A1 (fr) 1998-06-03 1998-06-03 Procédés d'essai, dispositif et marqueurs à base d'électrochimie

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WO2020180645A1 (fr) 2019-03-01 2020-09-10 Meso Scale Technologies, Llc. Sondes marquées par électrochimioluminescence destinées à être utilisées dans des procédés de dosage immunologique, procédés utilisant ces sondes et kits les comprenant
WO2020245377A1 (fr) 2019-06-07 2020-12-10 F. Hoffmann-La Roche Ag Hybridation d'oligonucléotides tout-lna
WO2021236584A1 (fr) 2020-05-19 2021-11-25 Meso Scale Technologies, Llc. Méthodes, compositions et kits de détection d'acides nucléiques
WO2022006236A1 (fr) 2020-07-01 2022-01-06 Meso Scale Technologies, Llc. Compositions et procédés pour mesures de dosage
WO2022136234A1 (fr) 2020-12-22 2022-06-30 F. Hoffmann-La Roche Ag Procédé de détection d'un analyte d'intérêt dans un échantillon
CN114371161B (zh) * 2021-11-15 2023-09-22 吉林大学 一种基于具有偏振分辨特性的表面增敏电化学发光分析方法及应用
CN114371161A (zh) * 2021-11-15 2022-04-19 吉林大学 一种基于具有偏振分辨特性的表面增敏电化学发光分析方法及应用
WO2023129884A1 (fr) 2021-12-30 2023-07-06 Meso Scale Technologies, Llc. Procédés de détection par électrochimiluminescence
WO2023156510A1 (fr) 2022-02-18 2023-08-24 F. Hoffmann-La Roche Ag Procédé de détection d'un analyte d'intérêt dans un échantillon

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WO1999063347A3 (fr) 2000-04-06
AU4372199A (en) 1999-12-20
EP0962773A1 (fr) 1999-12-08

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